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Globally, parasite-induced diseases in humans and wildlife are on the rise, and pesticide pollution may be a contributing factor. Echinostoma spp. trematode parasites are prominent in North America, and they use ramshorn snails (Planorbella [Helisoma] trivolvis) as intermediate hosts. We investigated the impact of chronic exposure to 1 of 5 pesticide treatments (control, or 50 μg/L of atrazine, glyphosate, carbaryl, or malathion) on uninfected and Echinostoma-infected snails for 41 d in the laboratory. We recorded snail mortality, the number of egg masses laid, change in mass, and behavior. Chronic exposure to atrazine, carbaryl, and malathion significantly decreased snail survival, whereas parasite infection status or exposure to glyphosate did not. Pesticide and parasite treatments did not influence growth or behavior, but parasite infection caused complete reproductive failure in snail hosts. Our results indicated that the direct effects of pesticides could threaten snail populations in natural environments and disrupt host-parasite dynamics. Environ Toxicol Chem 2021;40:2755-2763. © 2021 SETAC.

The current biodiversity crisis has intensified the need to predict species responses to landscape modification and has renewed attention on the fundamental question of what influences the dynamics of species distributions. Landscape composition can affect two main components that dictate distributions: habitat suitability and habitat connectivity. Elucidating the relative importance of these factors and associated landscape features can help prioritize management action for species conservation. Our objective was to use species distribution models and network-based landscape connectivity models to understand which landscape factors were most predictive of the distribution of an anuran, Blanchard’s cricket frog (Acris blanchardi), in an agriculturally-dominated landscape. We conducted our study in Ohio, USA, near the edge of the cricket frog’s contracting range. To obtain a current assessment of cricket frog distribution, we surveyed 367 pond and stream locations across three North–South transects. We then tested seven regression models, combining habitat suitability and landscape connectivity metrics, to determine which factors best predicted cricket frog presence. We detected cricket frogs in 24% of surveyed locations and they were more likely to occupy pond sites than stream sites. Cricket frog presence was best predicted by models with habitat suitability and the number of interconnected habitat patches. We found that, while there was high variation in habitat suitability across the study area, landscape connectivity was relatively uniform where we surveyed. Agricultural landscapes around the world are often mosaics of land cover types, which may functionally provide connectivity for some species. In such areas, conservation management should focus on preserving and restoring regions of highly suitable habitat. This focus may be particularly relevant for species that do not appear to be dispersal limited and, therefore, able to maintain metapopulation dynamics.

The role of parasites can change depending on the food web community. Predators, for instance, can amplify or dilute parasite effects on their hosts. Likewise, exposure to parasites or predators at one life stage can have long-term consequences on individual performance and survival, which can influence population and disease dynamics. To understand how predators affect amphibian parasite infections across life stages, we manipulated exposure of northern leopard frog (

Emerging infectious diseases are increasing globally and are an additional challenge to species dealing with native parasites and pathogens. Therefore, understanding the combined effects of infectious agents on hosts is important for species’ conservation and population management. Amphibians are hosts to many parasites and pathogens, including endemic trematode flatworms (e.g., Echinostoma spp.) and the novel pathogenic amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Our study examined how exposure to trematodes during larval development influenced the consequences of Bd pathogen exposure through critical life events. We found that prior exposure to trematode parasites negatively impacted metamorphosis but did not influence the effect of Bd infection on terrestrial growth and survival. Bd infection alone, however, resulted in significant mortality during overwintering—an annual occurrence for most temperate amphibians. The results of our study indicated overwintering mortality from Bd could provide an explanation for enigmatic declines and highlights the importance of examining the long-term consequences of novel parasite exposure.

We exposed Blanchard's cricket frog (Acris blanchardi) tadpoles to atrazine in simulated aquatic communities (outdoor mesocosms) at nominal concentrations of 0, 1, 10, 100, and 200 μg/L and tracked the effects of exposure to spring emergence in the laboratory, as well as to reproductive age in outdoor, terrestrial enclosures. We tested hypotheses that 1) atrazine addition increases the prevalence and intensity of testicular ova (TO) among phenotypic males at metamorphosis and after overwintering, 2) atrazine reduces maturation of ova after overwintering among phenotypic females, and 3) atrazine alters mass, time, and survival to metamorphosis, as well as growth and survival across terrestrial life stages. Atrazine addition increased probability of TO presence at metamorphosis, but only when treatments were pooled and compared to the control, where background atrazine was detected. Atrazine did not influence the intensity of TO among metamorphs. We observed TO among males at spring emergence and at reproductive age regardless of exposure concentration. We found no evidence for effects of exposure on gonadal maturation among females after overwintering. Exposure to 200 μg/L reduced survival to metamorphosis, but atrazine did not affect mass at metamorphosis, time to metamorphosis, or survival or mass after overwintering. We demonstrate that atrazine addition can increase TO prevalence relative to background rates at metamorphosis and that TO are also present among phenotypic males after overwintering. We suggest that this non-model species is sensitive to effects of larval EDC exposures on gonadal development and morphology and that further work with cricket frogs is warranted.

Chemical contamination of aquatic environments is widespread, but we have a limited understanding of how contaminants alter critical host–parasite interactions that can influence disease dynamics. We manipulated Northern Leopard Frog (Lithobates pipiens) exposure to pesticides (no pesticides, the insecticide Bacillus thuringiensis israelensis [Bti], or the herbicide atrazine) and trematode-infected (Ribeiroia ondatrae and Echinostoma spp.) snails in outdoor mesocosms. Bti exposure extended host larval period, and atrazine exposure had a nonsignificant trend toward reducing host survival; however, neither pesticide influenced parasite success nor magnified the effects of parasites on their hosts. Parasites negatively influenced tadpole development and, by metamorphosis, parasitized frogs had severe limb deformities and greater mass than unparasitized frogs. The greater mass in parasitized frogs may have resulted from reduced competition between tadpoles and snails for algal resources because parasites decreased snail abundance in mesocosms. Reduced competition between tadpoles and snails may offset the direct negative effects of trematodes on tadpoles, enabling them to survive with high infection intensities. Trematodes may further facilitate their own success by inducing limb deformities that likely increase anuran consumption by definitive hosts. Our results demonstrate how common pesticides and parasites impact amphibians and suggest that, at environmentally relevant concentrations, these pesticides may not dramatically alter host–parasite dynamics.

The destruction of freshwater habitat is a major contributor to biodiversity loss in aquatic ecosystems. However, created or restored wetlands could partially mitigate aquatic biodiversity loss by increasing the amount of available habitat across a landscape. We investigated the impact of surrounding terrestrial habitat and water quality variables on suitability for two species of pond-breeding amphibians (bullfrogs [Lithobates catesbeianus] and Blanchard’s cricket frogs [Acris blanchardi]) in created permanent wetlands located on an agricultural landscape. We examined tadpole growth and survival in field enclosures placed in ponds surrounded by agricultural, forested, or grassland habitats. We also evaluated the potential for carryover effects of the aquatic environment on terrestrial growth and overwinter survival of cricket frog metamorphs. We found that habitat adjacent to ponds did not predict tadpole growth or survival. Rather, phytoplankton abundance, which showed high variability among ponds within habitat type, was the only predictor of tadpole growth. Cricket frogs emerged larger and earlier from ponds with higher phytoplankton abundance; bullfrogs were also larger and at a more advanced developmental stage in ponds with higher levels of phytoplankton. Overwinter survival of cricket frogs was explained by size at metamorphosis and there were no apparent carryover effects of land use or pond-of-origin on overwinter growth and survival. Our results demonstrate that created ponds in human-dominated landscapes can provide suitable habitat for some anurans, independent of the adjacent terrestrial habitat.

Infectious pathogens threaten wildlife populations through effects on host growth, reproduction, and survival. The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), the cause of the disease chytridiomycosis, has been implicated in worldwide declines of amphibian populations. Documenting conditions under which amphibians are threatened by Bd will allow us to pinpoint at-risk populations, especially in the midwestern United States, an understudied region. We investigated how distributions of Bd differ across host life stages, land cover types, and spatial extents in a susceptible temperate host, the American Toad (Anaxyrus americanus). We surveyed a total of 232 adults at 14 sites and 430 metamorphs at 15 sites in southwestern Ohio, USA, and analyzed associations among land use type, Bd prevalence, and Bd load by using model selection. We found Bd infection prevalence in metamorphs was dramatically lower than in adults; only 1.6% of metamorphs were infected compared with 28.0% of adults. These results suggest that Bd transmission occurs after metamorphosis in this species or that infections vary with season, given that we surveyed adults in the spring during breeding events and metamorphs in the summer as they emerged from ponds. In adults, infection prevalence was reduced with increasing open-canopy habitats across spatial scales (from 100 to 1,000 m), whereas infection load increased with the proportion of forested habitats at small spatial scales (100 m). Our study shows that Bd infection risk in a temperate system can be influenced by host life stage and land cover types across local spatial scales.

In areas with heavy pesticide use, it is easy to attribute population declines to environmental contamination. The Blanchard's cricket frogs (Acris blanchardi) is an amphibian experiencing declines and range contractions across its distribution in the Midwest Corn Belt (USA). Experimental studies suggest that cricket frogs are sensitive to pesticides, but there are few studies examining this species' susceptibility to contaminants in realistic environments or comparing relative impacts with other anuran species. I reared 3 summer breeding anurans in outdoor mesocosms posthatching through metamorphosis to examine the effects of 2 insecticides (imidacloprid and carbaryl) and 1 herbicide (glyphosate with polyoxyethylene tallow amine) on larval development and metamorphosis. Cricket frogs were positively affected by insecticide exposure, likely a result of changes in the food web that increased food abundance. However, metamorphosis of green frogs (Lithobates clamitans) and gray tree frogs (Hyla chrysoscelis) appeared unaffected by pesticide exposure. The results of the present study suggest that the impacts of pesticides alone are unlikely to have population-level impacts for the anurans examined. Environ Toxicol Chem 2018;37:2699-2704. © 2018 SETAC.

We exposed Blanchard's cricket frogs (Acris blanchardi) to ecologically relevant concentrations (0, 0.1, 1, and 10 μg/L) of a commercial formulation of atrazine throughout the larval period to determine effects on survival, somatic growth and development (time to metamorphosis and mass at metamorphosis), and gonadal development (sex ratio at metamorphosis and the prevalence of testicular ova in phenotypic males). We tested the following hypotheses: 1) atrazine feminizes the sex ratio, 2) atrazine increases the proportion of phenotypic males with testicular ova, and 3) atrazine differentially affects somatic growth (mass at metamorphosis) and development (time to metamorphosis) for males and females. Although the control sex ratio was male-biased, exposure to 0.1 and 10 μg/L atrazine feminized sex ratios, because these treatments produced 51 and 55% fewer males than the control, respectively. We did not observe testicular ova. Atrazine did not impact survival or metamorphosis, and we did not detect sexually dimorphic impacts on time to metamorphosis or mass at metamorphosis. However, males metamorphosed 2.3 d later than females, regardless of treatment. Sex biases in timing of metamorphosis are underexplored in anurans, but if prevalent, could have important implications for theory surrounding the impact of environmental factors on metamorphosis. Our data suggest that cricket frog sex ratios are sensitive to environmentally relevant concentrations of atrazine and that feminization in the field is likely. Environ Toxicol Chem 2018;37:427-435. © 2017 SETAC.

Understanding factors that influence host–pathogen interactions is key to predicting outbreaks in natural systems experiencing environmental change. Many amphibian population declines have been attributed to an amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). While this fungus is widespread, not all Bd‐positive populations have been associated with declines, which could be attributed to differences in pathogen virulence or host susceptibility. In a laboratory experiment, we examined the effects of Bd isolate origin, two from areas with Bd‐associated amphibian population declines (El Copé, Panama, and California, USA) and two from areas without Bd‐related population declines (Ohio and Maine, USA), on the terrestrial growth and survival of American toad (Anaxyrus americanus) metamorphs reared in larval environments with low or high intraspecific density. We predicted that (1) Bd isolates from areas experiencing declines would have greater negative effects than Bd isolates from areas without declines, and (2) across all isolates, growth and survival of smaller toads from high‐density larval conditions would be reduced by Bd exposure compared to larger toads from low‐density larval conditions. Our results showed that terrestrial survival was reduced for smaller toads exposed to Bd with variation in the response to different isolates, suggesting that smaller size increased susceptibility to Bd. Toads exposed to Bd gained less mass, which varied by isolate. Bd isolates from areas with population declines, however, did not have more negative effects than isolates from areas without recorded declines. Most strikingly, our study supports that host condition, measured by size, can be indicative of the negative effects of Bd exposure. Further, Bd isolates’ impact may vary in ways not predictable from place of origin or occurrence of disease‐related population declines. This research suggests that amphibian populations outside of areas experiencing Bd‐associated declines could be impacted by this pathogen and that the size of individuals could influence the magnitude of Bd's impact.

Studies of endocrine-disrupting contaminants have focused on early-life exposures, but later exposures could impact fitness. We exposed adult frogs (Acris blanchardi) at reproduction to ecologically relevant atrazine concentrations (0, 1, or 10 µg/L) in outdoor arenas. We measured likelihood of breeding and number of resulting tadpoles. Atrazine impacted neither the probability of breeding nor the number of tadpoles produced, suggesting anuran reproductive success may not be impacted by short-term exposure to low concentrations. Environ Toxicol Chem 2017;36:3284-3288. © 2017 SETAC.

Host species may differ in their responses to pathogen exposures based on host energy reserves, which could be important for long-term trends in host population growth.

Exurban development is a prominent land use in the United States of America, particularly in the Midwest, where much of it occurs on farmland and remnant woodlands. While exurbanization may affect ecosystem services, its impact could be modulated by management decisions made by residents. We aimed to uncover how exurban residents in a midwestern county perceived ecosystem services and disservices provided by their property based on 26 semi-structured interviews of landowners on parcels between 1 and 20 acres with a pond in unincorporated areas. We found the ecosystem services people associated most with their land are classified as cultural services (dominated by recreation services), while the most common mentioned disservices were classified as regulating disservices. Many ecosystem services that would be categorized as supporting or regulating services were not mentioned by interviewees, including microclimate stabilization, carbon sequestration, disease regulation, and maintenance of genetic diversity. Residents spent an average of 1.4 h/acre each week managing their properties. However, as parcel size and forest cover increased, the residents reported managing less surface area. Our study suggested that residents cultivate landscape features that directly benefit them and view many of the services that benefit regional biodiversity and ecosystem processes as disservices, which, to rectify, may require coordinated landscape-level management or local policies/incentives.

Intraspecific variability in contaminant sensitivity could undermine risk assessments for nontarget organisms such as amphibians. To test how amphibian populations vary in tolerance to anticipated lethal and sublethal exposures to a pesticide, we exposed Blanchard's cricket frogs (Acris blanchardi) from 3 populations across a broad portion of their range to the insecticide malathion. Exposure in mesocosms to a nominal concentration of 1 mg/L (measured concentrations at 1 h and 24 h postaddition of 0.160 mg/L and 0.062 mg/L, respectively), a realistic direct-overspray scenario, reduced survival to metamorphosis by 43% relative to controls and revealed variation in tolerance among populations. Survival ranged from 74% for the most tolerant population to 18% for the least tolerant population, a 4.1-fold difference. Mass at metamorphosis and time to metamorphosis were unaffected. Although malathion reduced zooplankton abundance, it did not alter food resources (periphyton or phytoplankton relative abundance), or a suite of water-quality variables (pH, temperature, and dissolved oxygen). A 96-h time-to-death assay designed to isolate direct, lethal effects also revealed variation in tolerance among populations. Time to death (mean ± standard error) ranged from 2.4 ± 0.18 h for the least tolerant population to 17.8 ± 4.72 h for the most tolerant population, a 7.4-fold difference. However, relative sensitivities of populations differed in the mesocosm and laboratory studies, which differed in exposure concentrations, suggesting that populations tolerant of high concentrations can be more sensitive to lower concentrations. We suggest that direct overspray could reduce larval survival in the field for this species. Studies assessing the role of contaminants in declines or extrapolating to untested populations, especially across large geographical regions, should quantify the range of intraspecific variation. Risk assessors could address intraspecific variability directly by using an intraspecific uncertainty factor. Environ Toxicol Chem 2017;36:1917-1923. © 2016 SETAC.

Species distributions are a function of an individual’s ability to disperse to and colonize habitat patches. These processes depend upon landscape configuration and composition. Using Blanchard’s cricket frogs (Acris blanchardi), we assessed which land cover types were predictive of (1) presence at three spatial scales (pond-shed, 500 and 2500 m) and (2) genetic structure. We predicted that forested, urban, and road land covers would negatively affect cricket frogs. We also predicted that agricultural, field, and aquatic land covers would positively affect cricket frogs. We surveyed for cricket frogs at 28 sites in southwestern Ohio, USA to determine presence across different habitats and analyze genetic structure among populations. For our first objective, we examined if land use (crop, field, forest, and urban habitat) and landscape features (ponds, streams, and roads) explained presence; for our second objective, we assessed whether these land cover types explained genetic distance between populations. Land cover did not have a strong influence on cricket frog presence. However, multiple competing models suggested effects of roads, streams, and land use. We found genetic structuring: populations were grouped into five major clusters and nine finer-scale clusters. Highways were predictive of increased genetic distance. By combining a focal-patch study with landscape genetics, our study suggests that major roads and waterways are key features affecting species distributions in agricultural landscapes. We demonstrate that cricket frogs may respond to landscape features at larger spatial scales, and that presence and movement may be affected by different environmental factors.

Pathogens can have a range of effects on organisms across environmental gradients; although lethal effects may receive greater attention, sublethal impacts can have important population- and community-level impacts. In this laboratory study, we examined the effects of exposure to the amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]) on Northern Leopard Frog (Lithobates pipiens) metamorphs that had been reared in the presence or absence of crayfish (Orconectes rusticus) predators in aquatic environments to determine whether prior predator exposure influenced the effect of Bd on terrestrial growth and survival of frogs. Although Bd exposure following metamorphosis did not impact Northern Leopard Frog survival, exposure did significantly reduce terrestrial growth, indicating that Bd exposure can have consequences that can indirectly impact populations, because size of amphibians is positively correlated with overwinter survival and fecundity. Exposure to predators during larval developm...

Pond hydroperiod and predators play a central role in structuring aquatic communities. Because of predicted changes in precipitation and temperature patterns associated with climate change, pond hydroperiods will likely be altered. Reduced hydroperiods can impact amphibian populations by restricting the amount of time available for larval growth and by altering predatory interactions via increased predator densities. We investigated how pond drying and predation singularly and interactively affected growth and survival of Acris blanchardi (Blanchard's Cricket Frogs). We reared recently hatched tadpoles through metamorphosis in outdoor mesocosms using a factorial design incorporating three hydroperiods (fast-drying, slow-drying, or constant) and three larval odonate predator treatments (caged, uncaged, or absent). Caged and uncaged predator treatments were implemented to evaluate both consumptive and non-consumptive effects. There were no differences in survival, time to metamorphosis, or size at metamorph...

Host species may differ in their responses to pathogen exposures based on host energy reserves, which could be important for long-term trends in host population growth. Batrachochytrium dendrobatidis (BD) is a pathogen associated with amphibian population declines but also occurs without causing mass mortalities. The impact of BD in populations without associated declines is not well understood, and food abundance could play a role in determining the magnitude of its effects. We exposed American toad (Anaxyrus americanus), northern leopard frog (Lithobates pipiens), and cricket frog (Acris blanchardi) metamorphs to BD under low or high food treatments. Overall, anuran species responded differently to BD exposure and the combined effect of BD exposure and food abundance was additive. American toad survival was lowered by BD exposure and low food availability. Based on these results, we developed a population model for American toads to estimate how reductions in survival could influence population growth. We found that BD could reduce population growth by 14% with high food availability and 21% with low food availability. In contrast, survival of northern leopard frogs was high across all treatments, but their growth was negatively impacted by the additive effects of BD exposure and low food availability. Cricket frog growth and survival were unaffected by BD exposure, suggesting that this species is not sensitive to the effects of this pathogen in terms of growth and survival across environments of different quality in the time period examined. Our results showed that low food availability additively increased the species-specific lethal and sublethal impacts of BD on hosts, which could have implications for long-term host population dynamics.

The severity of the impacts of pathogens on hosts may be driven by environmental factors like resource availability that create tradeoffs on energetic demands for immune responses and basal metabolic activity within the host. These responses can vary among species from sublethal to lethal effects, which can have consequences for the host population trajectories within a community. Chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis (or Bd), has been associated with global amphibian population declines. However, it also occurs in populations without appearing to cause mass mortality; the effect of Bd in these situations is not well understood and environmental factors like food abundance that impact host conditions could play an important role in the magnitude of the pathogen’s impact. In the present study, we exposed American toad (Anaxyrus americanus), northern leopard frog (Lithobates pipiens), and Blanchard’s cricket frog (Acris blanchardi) metamorphs to Bd and then reared them in the terrestrial habitat under low or high food environments. We found additive effects of Bd and reduced food abundance on host growth and survival that varied according to species. For instance, Bd-induced reductions in American toad survival were greater under low food conditions compared to high food conditions but survival of northern leopard frogs and Blanchard’s cricket frogs was not affected by Bd. For northern leopard frogs and Blanchard’s cricket frogs, low food abundance resulted in the lower growth rates under Bd exposure compared to high food abundance. Additionally, we developed stage-structured population models for American toads to assess if reduced survival of metamorphs exposed to Bd under conditions of low and high food abundance could influence population trajectories; models indicated that Bd exposure would reduce annual population growth rates by 14% under conditions of high food abundance and 21% under conditions of low food abundance. Our results suggest that environmental conditions that influence resource availability for species that are sensitive or tolerant to Bd may increase the negative effects of Bd on host growth and survival, which could have important implications for how populations and communities with infected members respond over time.

Effects of pathogens on host life-history characteristics can be influenced by changes in the immune system that occur during development as well as by environmental factors that negatively affect immune system function. Amphibians worldwide are experiencing population declines from chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis. Disease outbreaks can be influenced by timing of exposure to B. dendrobatidis and by abiotic factors, such as pesticides, that could influence susceptibility. To examine the effects of larval pesticide exposure and timing of B. dendrobatidis exposure during development, we exposed Cope’s Gray Treefrogs (Hyla chrysoscelis) to the insecticide malathion throughout larval development and to B. dendrobatidis at 1 or 3 wk posthatching, or after metamorphosis. We reared tadpoles through metamorphosis and then for 28 d in the terrestrial environment to examine treatment effects on larval survival, time to metamorphosis, and mass at metamorphosis,...

Mesocosms are an important tool in experimental aquatic ecology, but have been criticized for failing to effectively mimic natural habitats. Identifying contrived features of mesocosms that affect endpoints of interest is a prudent step in ensuring the reliability of mesocosm data. Because anuran larvae actively regulate their exposure to a suite of biotic and abiotic conditions by positioning themselves at various depths in natural ponds, the steep walls and minimal access to shallow regions in common cattle tank mesocosms may force tadpoles into sub-optimal patterns of habitat use. We tested whether adding angled ramps or horizontal platforms to increase access to shallow regions affected survival, time, or mass at metamorphosis of American Toads (Anaxyrus americanus) or Northern Leopard Frogs (Lithobates pipiens) in the presence or absence of Rusty Crayfish (Orconectes rusticus), a common predator that may influence habitat use. Addition of structure had no effect on American Toad survival or metamorph...

In areas with heavy pesticide use, it is easy to attribute population declines to environmental contamination. The Blanchard's cricket frogs (Acris blanchardi) is an amphibian experiencing declines and range contractions across its distribution in the Midwest Corn Belt (USA). Experimental studies suggest that cricket frogs are sensitive to pesticides, but there are few studies examining this species' susceptibility to contaminants in realistic environments or comparing relative impacts with other anuran species. I reared 3 summer breeding anurans in outdoor mesocosms posthatching through metamorphosis to examine the effects of 2 insecticides (imidacloprid and carbaryl) and 1 herbicide (glyphosate with polyoxyethylene tallow amine) on larval development and metamorphosis. Cricket frogs were positively affected by insecticide exposure, likely a result of changes in the food web that increased food abundance. However, metamorphosis of green frogs (Lithobates clamitans) and gray tree frogs (Hyla chrysoscelis) appeared unaffected by pesticide exposure. The results of the present study suggest that the impacts of pesticides alone are unlikely to have population-level impacts for the anurans examined. Environ Toxicol Chem 2018;37:2699-2704. © 2018 SETAC.

Abiotic factors such as pesticides may alter the impact of a pathogen on hosts, which could have implications for host–pathogen interactions and may explain variation in disease outbreaks in nature. In the present laboratory experiment, American toad (Anaxyrus americanus) metamorphs were exposed to the amphibian chytrid fungal pathogen Batrachochytrium dendrobatidis (Bd) and environmentally relevant concentrations of the insecticide malathion to determine whether malathion altered the effects of Bd exposure on growth and survival of toad metamorphs. Exposure to Bd significantly decreased survival over the 51 d of the experiment, suggesting that Bd could reduce recruitment into the terrestrial life stage when exposure occurs at metamorphosis. Malathion did not impact survival, but a 12-h exposure at metamorphosis significantly reduced terrestrial growth. Toads that were exposed to both Bd and malathion showed a nonsignificant trend toward the smallest growth compared with other treatments. The present study suggests that Bd may pose a threat to American toads even though population declines have not been observed for this species; in addition, the presence of both the insecticide malathion and Bd could reduce terrestrial growth, which could have implications for lifetime fitness and suggests that environmental factors could play a role in pathogen impacts in nature. Environ Toxicol Chem 2014;33:2541–2544. © 2014 SETAC

Chytridiomycosis, a disease caused by Batrachochytrium dendrobatidis (Bd), has been implicated as a cause of amphibian declines. Susceptibility may be influenced by environmental factors that suppress the immune response. The authors conducted a laboratory study to examine the effect of temperature, insecticide exposure, and Bd exposure during larval anuran development. The authors examined the consequences of exposure to Bd, an insecticide (carbaryl or malathion), and static or fluctuating temperature (15 °C, 20 °C, 25 °C, or 15 °C to 25 °C 72-h flux) on larval development through metamorphosis of the Pacific treefrog (Pseudacris regilla). High and fluctuating temperature had negative effects on survival in the presence of Bd. Insecticides inhibited the effects of Bd; time to tail resorption of Pacific treefrogs decreased when tadpoles were exposed to carbaryl. The present study indicates that abiotic factors may play a role in the host–pathogen interactions in this system. Environ Toxicol Chem 2014;33:2545–2550. © 2014 SETAC

Pesticide use results in the widespread distribution of chemical contaminants, which necessites regulatory agencies to assess the risks to environmental and human health. However, risk assessment is compromised when relatively few studies are used to determine impacts, particularly if most of the data used in an assessment are produced by a pesticide’s manufacturer, which constitutes a conflict of interest. Here, we present the shortcomings of the US Environmental Protection Agency’s pesticide risk assessment process, using the recent reassessment of atrazine’s impacts on amphibians as an example. We then offer solutions to improve the risk assessment process, which would reduce the potential for and perception of bias in a process that is crucial for environmental and human health.Keywords: amphibian, atrazine, conflicts of interest, Environmental Protection Agency, risk assessment

Pesticides are detectable in most aquatic habitats and have the potential to alter host-pathogen interactions. The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), has been associated with amphibian declines around the world. However, Bd-associated declines are more prominent in some areas, despite nearly global distribution of Bd, suggesting other factors contribute to disease outbreaks. In a laboratory study, the authors examined the effects of 6 different isolates of Bd in the presence or absence of a pesticide (the insecticide carbaryl or the fungicide copper sulfate) to recently hatched Cope's gray treefrog (Hyla chrysoscelis) tadpoles reared through metamorphosis. The authors found the presence or absence of pesticides differentially altered the mass at metamorphosis of tadpoles exposed to different Bd isolates, suggesting that isolate could influence metamorphosis but not in ways expected based on origin of the isolate. Pesticide exposure had the strongest impact on metamorphosis of all treatment combinations. Whereas copper sulfate exposure reduced the length of the larval period, carbaryl exposure had apparent positive effects by increasing mass at metamorphosis and lengthening larval period, which adds to evidence that carbaryl can stimulate development in counterintuitive ways. The present study provides limited support to the hypothesis that pesticides can alter the response of tadpoles to isolates of Bd and that the insecticide carbaryl can alter developmental decisions.

Long-term population persistence depends on successful dispersal and colonization. Within agricultural landscapes, dispersing individuals encounter a variety of edge types. How individuals respond to edges can dictate whether they are permeable to dispersal or act as barriers, with consequences for population connectivity. Using two amphibian species, bullfrogs ( Lithobates catesbeianus ) and Blanchard’s cricket frogs ( Acris blanchardi ), we conducted two studies to address (1) how abiotic factors influence habitat choice and (2) how habitat choice and movement behavior change based on edge type (edges were between grass–corn, grass–forest, and forest–corn habitats). In the first experiment we found that both species preferred high soil moisture environments during both the day and night; and at night neither species showed a preference for ground or canopy cover. However, during the day bullfrogs had a preference for both ground and canopy cover, while cricket frogs showed no preference. In the second experiment, we found that bullfrogs had no overall preference for one habitat type over another; they were observed traveling along the edge of two habitats. Cricket frogs, on the other hand, showed a strong aversion to forest habitat. We concluded that behavioral responses to different habitats may explain current population trends; movement along edge habitat may allow bullfrogs to readily disperse through altered landscapes whereas cricket frog declines may occur in areas experiencing reforestation due to increased isolation. Conservation practices in agricultural land will likely improve landscape connectivity for these species, especially for declining cricket frog populations.

Understanding the effects of pesticide exposure on organisms throughout their life cycle is critical to predict population-level effects. For many taxa, including amphibians, juveniles are the main dispersal stage and are disproportionally important to population persistence when compared with other life stages. In the present study, we examined the effects of a single 24-h exposure to the insecticide carbaryl or the herbicide atrazine on locomotor performance (endurance, distance traveled, speed, and fatigue) in the laboratory and terrestrial growth and survival through overwintering in field enclosures using recent metamorphs of spotted salamanders (Ambystoma maculatum). We found that neither atrazine nor carbaryl impacted endurance, but fatigue increased with carbaryl exposure, which could leave salamanders less able to escape repeated attacks by predators. Terrestrial growth and overwinter survival were not affected by short-term exposure to carbaryl or atrazine, suggesting that when individuals can overcome acute effects, no long-term consequences result for the endpoints measured. Environ Toxicol Chem 2014;33:548–552. © 2013 SETAC

We exposed Blanchard's cricket frogs (Acris blanchardi) to ecologically relevant concentrations (0, 0.1, 1, and 10 μg/L) of a commercial formulation of atrazine throughout the larval period to determine effects on survival, somatic growth and development (time to metamorphosis and mass at metamorphosis), and gonadal development (sex ratio at metamorphosis and the prevalence of testicular ova in phenotypic males). We tested the following hypotheses: 1) atrazine feminizes the sex ratio, 2) atrazine increases the proportion of phenotypic males with testicular ova, and 3) atrazine differentially affects somatic growth (mass at metamorphosis) and development (time to metamorphosis) for males and females. Although the control sex ratio was male-biased, exposure to 0.1 and 10 μg/L atrazine feminized sex ratios, because these treatments produced 51 and 55% fewer males than the control, respectively. We did not observe testicular ova. Atrazine did not impact survival or metamorphosis, and we did not detect sexually dimorphic impacts on time to metamorphosis or mass at metamorphosis. However, males metamorphosed 2.3 d later than females, regardless of treatment. Sex biases in timing of metamorphosis are underexplored in anurans, but if prevalent, could have important implications for theory surrounding the impact of environmental factors on metamorphosis. Our data suggest that cricket frog sex ratios are sensitive to environmentally relevant concentrations of atrazine and that feminization in the field is likely. Environ Toxicol Chem 2018;37:427-435. © 2017 SETAC.

Studies of endocrine-disrupting contaminants have focused on early-life exposures, but later exposures could impact fitness. We exposed adult frogs (Acris blanchardi) at reproduction to ecologically relevant atrazine concentrations (0, 1, or 10 µg/L) in outdoor arenas. We measured likelihood of breeding and number of resulting tadpoles. Atrazine impacted neither the probability of breeding nor the number of tadpoles produced, suggesting anuran reproductive success may not be impacted by short-term exposure to low concentrations. Environ Toxicol Chem 2017;36:3284-3288. © 2017 SETAC.

Intraspecific variability in contaminant sensitivity could undermine risk assessments for nontarget organisms such as amphibians. To test how amphibian populations vary in tolerance to anticipated lethal and sublethal exposures to a pesticide, we exposed Blanchard's cricket frogs (Acris blanchardi) from 3 populations across a broad portion of their range to the insecticide malathion. Exposure in mesocosms to a nominal concentration of 1 mg/L (measured concentrations at 1 h and 24 h postaddition of 0.160 mg/L and 0.062 mg/L, respectively), a realistic direct-overspray scenario, reduced survival to metamorphosis by 43% relative to controls and revealed variation in tolerance among populations. Survival ranged from 74% for the most tolerant population to 18% for the least tolerant population, a 4.1-fold difference. Mass at metamorphosis and time to metamorphosis were unaffected. Although malathion reduced zooplankton abundance, it did not alter food resources (periphyton or phytoplankton relative abundance), or a suite of water-quality variables (pH, temperature, and dissolved oxygen). A 96-h time-to-death assay designed to isolate direct, lethal effects also revealed variation in tolerance among populations. Time to death (mean ± standard error) ranged from 2.4 ± 0.18 h for the least tolerant population to 17.8 ± 4.72 h for the most tolerant population, a 7.4-fold difference. However, relative sensitivities of populations differed in the mesocosm and laboratory studies, which differed in exposure concentrations, suggesting that populations tolerant of high concentrations can be more sensitive to lower concentrations. We suggest that direct overspray could reduce larval survival in the field for this species. Studies assessing the role of contaminants in declines or extrapolating to untested populations, especially across large geographical regions, should quantify the range of intraspecific variation. Risk assessors could address intraspecific variability directly by using an intraspecific uncertainty factor. Environ Toxicol Chem 2017;36:1917-1923. © 2016 SETAC.

Habitat structure is known to influence community interactions, but its role in amphibian communities is unclear. Our objective was to examine the effect of vegetative habitat structure, in the presence or absence of a crayfish predator (Orconectes rusticus) and the herbicide atrazine, on Green Frog (Rana clamitans) tadpoles reared in 10-L microcosms in the laboratory. Crayfish predators reduced Green Frog survival and affected activity levels and microhabitat use differently depending on the presence or absence of vegetation. In treatments with vegetation, activity levels were greater when the crayfish predator was absent, but in treatments without vegetation activity levels did not differ between predation treatments. Moreover, when a crayfish predator was present, tadpoles in treatments without vegetation spent more time at the water surface than tadpoles in treatments with vegetation. This preference for the water surface may have been a compensatory behavioral change to avoid predation becau...

Chemical contamination may influence host-pathogen interactions, which has implications for amphibian population declines. We examined the effects of four insecticides alone or as a mixture on development and metamorphosis of Pacific Treefrogs (Pseudacris regilla) in the presence or absence of the amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Bd exposure had a negative impact on tadpole activity, survival to metamorphosis, time to metamorphosis, and time of tail absorption (with a marginally negative effect on mass at metamorphosis); however, no individuals tested positive for Bd at metamorphosis. The presence of sublethal concentrations of insecticides alone or in a mixture did not impact Pacific Treefrog activity as tadpoles, survival to metamorphosis, or time and size to metamorphosis. Insecticide exposure did not influence the effect of Bd exposure. Our study did not support our prediction that effects of Bd would be greater in the presence of expected environmental concentr...

Northern Cricket Frogs, Acris crepitans, are experiencing population declines throughout their range for unknown reasons. Habitat alteration is one potential explanation, so evaluating habitat use and movement of Cricket Frogs could be valuable to protect and manage this species. To understand how altered landscapes influence Cricket Frog movements, we initiated a study at a golf course to assess the dispersal of adults on a variety of terrestrial habitat types (mown grass, unmown grass, or the ecotone between mown and unmown grass) released at different distances from a single pond (10, 20, or 40 m). We monitored movements of adult male frogs by marking individuals with fluorescent powder. Distance from the pond did not appear to affect movement or orientation toward the pond. However, Cricket Frog movement was significantly affected by habitat type. Cricket Frogs produced longer paths in unmown grass and were more likely to orient toward the pond than frogs released at the ecotone, which may be benefici...

Priority effects are defined as asymmetric responses of different species colonizing the same habitat in different sequences and are important in structuring communities, particularly for ephemeral systems that are colonized annually. Amphibians often use ephemeral ponds and have been shown to experience priority effects from interspecific competition. Pesticide exposure can impact amphibian species interactions like competition, and has been linked to declines. We investigated whether insecticide exposure interacted with colonization history to impact priority effects in Bufo americanus and Rana pipiens. We predicted that exposure to the insecticide carbaryl and colonization history would affect tadpole metamorphosis independently and interactively. Our results indicated that Bufo time to metamorphosis experienced priority effects: Bufo introduced early reached metamorphosis sooner when Rana were absent, but Bufo introduced late reached metamorphosis sooner when Rana were also late. Insecticide exposure eliminated differences in Bufo time to metamorphosis due to Rana colonization history. Rana survival and mass at metamorphosis were affected by intraspecific colonization history. Bufo had significantly fewer survivors and Rana had more survivors in insecticide-exposed treatments. Bufo did better when reared without Rana, but Rana was unaffected by Bufo presence. The effects on anuran metamorphosis occurred without any effects on periphyton abundance. We suggest that the effects of insecticide exposure on amphibian metamorphosis are not related to colonization history, although each factor alone can have substantial impacts. These results preliminarily suggest that managing pesticide applications with sensitivity to amphibians may be less complex than implied by traditional food web theory, although more research is needed.

Worldwide amphibian population declines have occurred in the last few decades and have been attributed to a range of factors including introduced species and chemical contamination. Anuran species may differ in their susceptibility to declines based on life-history characteristics, leading to different probabilities of decline and conservation statuses. In this experiment, we looked at two anuran species, Northern Cricket Frogs (Acris crepitans) and Green Frogs (Rana clamitans), reared in mesocosms containing a common invasive or introduced potential predator (Rusty Crayfish, Bluegill Sunfish, or triploid Grass Carp) and imidacloprid, a common insecticide. We found that anurans differed in their sensitivity to these factors. Cricket Frog survival was significantly reduced with imidacloprid exposure, whereas Green Frogs were not. Abundance of both amphibian species was reduced in the presence of predators, particularly the fish. Our study suggests that Cricket Frogs may be especially sensitive to the insecticide imidacloprid, as well as fish predators, and that these factors could contribute to their population declines.

Commercial formulations of pesticides contain both active and other ingredients. In some instances, the other ingredients have detrimental effects on nontarget species. Other factors such as UV radiation and predator cues have been shown to modify the toxicity of pesticides. In a laboratory study we compared the effects of technical-grade active ingredients to commercial formulations of seven common pesticides in the presence or absence of UV radiation on the survival of Rana clamitans (green frog) tadpoles over 96 h. We found a significant difference in the survival of tadpoles in technical-grade active ingredients versus commercial formulations in all of the pesticides tested. We also found that either the presence or the absence of UV radiation affected the survival of tadpoles in five of the seven pesticides tested. These results suggest that there is a need to test the effects of both active ingredients and commercial formulations of pesticides and, also, to include relevant abiotic factors like UV radiation treatments in the testing of pesticides because they can have a dramatic impact on the toxicity of some chemicals.

Many research studies have focused on the interactions among larval anurans, but relatively little is known about competitive or predatory interactions among anurans following metamorphosis. The purpose of our study was to examine the effects of post-metamorphic green frogs (Rana clamitans) on post-metamorphic American toads (Bufo americanus) in the terrestrial environment as a model for understanding how interspecific differences in size at metamorphosis may influence terrestrial interactions. We examined if caged, uncaged or no green frogs affected survival, growth and hiding behavior of recently metamorphosed American toads in a replicated laboratory experiment. Green frogs did not appear to prey upon small American toads. However, by the end of the experiment American toad survival was lowest in treatments with uncaged green frogs; this result suggests that green frogs may outcompete American toads for food, but such impacts in nature may be limited or may have selected for spatial segregation in the terrestrial environment. Green frogs also increased hiding activity of toads at some times, which may in part explain the trend of reduced mass gained when toads were reared with green frogs. This study represents one of the first to examine interspecific interactions among anurans with complex life cycles in the terrestrial environment.

Although the number of temporary wetlands used by many amphibian species has declined nationally, permanent wetlands have increased on the landscape in many regions. Species like Bullfrogs (Rana catesbeiana) may benefit from permanent wetlands and increase their density on the landscape, making them more likely to encounter wetlands used by amphibians breeding in temporary ponds. Although Bullfrogs are typically viewed as inhabitants of permanent wetlands because of their long larval periods, they are known to use temporary ponds. This study examined how larval density influenced proportion of Bullfrogs metamorphosing, time to metamorphosis, mass at metamorphosis, and total Bullfrog survival in mesocosm ponds. Proportion of Bullfrogs metamorphosing and tadpole development were greatest in mesocosms with low tadpole density with up to 25% of tadpoles reaching metamorphosis. Our study indicates Bullfrogs can metamorphose in more northern climates within a single season across a range of densities, and highlights the potential for Bullfrogs to successfully use temporary pond environments.

Managing areas designed for human recreation so that they are compatible with natural amphibian populations can reduce the negative impacts of habitat destruction. We examined the potential for amphibians to complete larval development in golf course ponds in the presence or absence of overwintered bullfrog tadpoles (Rana catesbeiana), which are frequently found in permanent, human-made ponds. We reared larval American toads (Bufo americanus), southern leopard frogs (R. sphenocephala), and spotted salamanders (Ambystoma maculatum) with 0 or 5 overwintered bullfrog tadpoles in field enclosures located in ponds on golf courses or in experimental wetlands at a reference site. Survival to metamorphosis of American toads, southern leopard frogs, and spotted salamanders was greater in ponds on golf courses than at reference sites. We attributed this increased survival to low abundance of insect predators in golf course ponds. The presence of overwintered bullfrogs, however, reduced the survival of American toads, southern leopard frogs, and spotted salamanders reared in golf course ponds, indicating that the suitability of the aquatic habitats for these species partly depended on the biotic community present. Our results suggest that ponds in human recreational areas should be managed by maintaining intermediate hydroperiods, which will reduce the presence of bullfrog tadpoles and predators, such as fish, and which may allow native amphibian assemblages to flourish.

A leading hypothesis of amphibian population declines is that combinations of multiple stressors contribute to declines. We examined the role that chemical contamination, competition, and predation play singly and in combination in aquatic amphibian communities. We exposed larvae of American toads (Bufo americanus), southern leopard frogs (Rana sphenocephala), and spotted salamanders (Ambystoma maculatum) to overwintered bullfrog tadpoles (R. catesbeiana), bluegill sunfish (Lepomis macrochirus), the insecticide carbaryl, and ammonium nitrate fertilizer in 1000-L mesocosms. Most significantly, our study demonstrated that the presence of multiple factors reduced survival of B. americanus and A. maculatum and lengthened larval periods of R. sphenocephala. The presence of bluegill had the largest impact on the community; it eliminated B. americanus and A. maculatum and reduced the abundance of R. sphenocephala. Chemical contaminants had the second strongest effect on the community with the insecticide, reducing A. maculatum abundance by 50% and increasing the mass of anurans (frogs and toads) at metamorphosis; the fertilizer positively influenced time and mass at metamorphosis for both anurans and A. maculatum. Presence of overwintered bullfrogs reduced mass and increased time to metamorphosis of anurans. While both bluegill and overwintered bullfrog tadpoles had negative effects on the amphibian community, they performed better in the presence of one another and in contaminated habitats. Our results indicate that predicting deleterious combinations from single-factor effects may not be straightforward. Our research supports the hypothesis that combinations of factors can negatively impact some amphibian species and could contribute to population declines.

In recent decades, created ponds have become one of the dominant aquatic habitats in anthropogenic landscapes. Understanding how competition between colonizing species influences community assembly in these new habitats is important for predicting species distributions across the landscape. The objective of this study was to examine competition between larval Cricket Frogs (Acris blanchardi), which are showing declines in parts of their range, and other anurans including Bullfrogs (Lithobates catesbeianus), Green Frogs (Lithobates clamitans), and Cope’s Gray Treefrogs (Hyla chrysoscelis). Using two separate experiments, we examined competitive interactions between larvae of (1) Cricket Frogs and different size classes of Bullfrogs; and (2) Cricket Frogs and Green Frogs, Cricket Frogs and Cope’s Gray Treefrogs, or all three species together. We found no effect of either recently hatched or overwintered Bullfrog larvae on Cricket Frog metamorphosis. However, we found that Green Frogs reduced Cric...

I reared four species of anurans (Rana sphenocephala [Southern Leopard Frog], Rana blairi [Plains Leopard Frog], Rana clamitans [Green Frog], and Bufo woodhousii [Woodhouse's Toad]) for seven to 12 months in small, outdoor terrestrial enclosures (1 × 2 m) to examine the consequences of larval competition (via density) and contaminant exposure (via the insecticide carbaryl). I added six Rana clamitans, eight Rana sphenocephala, eight Rana blairi, and 10 Bufo woodhousii to terrestrial enclosures shortly after metamorphosis and recaptured them during the following spring. All anurans from low-density ponds were significantly larger than those from high-density ponds, but these size differences did not significantly affect survival to or size at spring emergence. However, R. sphenocephala, R. blairi, and R. clamitans that survived to spring had been larger at metamorphosis on average than those that did not survive; in contrast, B. woodhousii that survived the winter were smaller at metamorphosis on ...

Aquatic and terrestrial mesocosms have been used successfully to examine ecological and ecotoxicological questions with amphibians. Mesocosms represent a bridge between the laboratory and the natural world, and allow for experimental research on factors that influence amphibian populations and communities. In this manuscript, we review the current literature utilizing mesocosms for ecotoxicology, evaluate the role that mesocosm studies could play in ecotoxicology, and suggest future research directions. Mesocosms are advantageous relative to other experimental systems because treatments are easily replicated, multiple environmental factors can be manipulated, food webs can be established, direct and indirect effects can be examined, and contamination effects can be evaluated. This allows for the examination of contaminant effects on multiple trophic and taxonomic levels in an ecologically relevant context. Standard guidelines for mesocosm testing with amphibians should be created to aid new researchers, make studies more comparable, and facilitate risk assessment.

We examined the interactive effects of overwintered Bullfrog (Rana catesbeiana) tadpoles and pond hydroperiod on a community of larval amphibians in outdoor mesocosms including American Toads (Bufo americanus), Southern Leopard Frogs (Rana sphenocephala), and Spotted Salamanders (Ambystoma maculatum)—species within the native range of Bullfrogs. Spotted Salamanders and Southern Leopard Frogs were negatively influenced by the presence of overwintered Bullfrogs. Spotted Salamanders had shorter larval periods and slightly smaller masses at metamorphosis, and Southern Leopard Frogs had smaller masses at metamorphosis when reared with Bullfrogs than without. Presence of overwintered Bullfrogs, however, did not significantly affect American Toads. Longer pond hydroperiods resulted in greater survival, greater size at metamorphosis, longer larval periods, and later time until emergence of the first metamorphs for Southern Leopard Frog tadpoles and Spotted Salamander larvae. Our study demonstrated that o...

We examined the effects of the insecticide carbaryl on larval amphibian communities in large-scale experimental ponds. Tadpoles of two anurans, Woodhouse's toad (Bufo woodhousii) and southern leopard frog (Rana sphenocephala), were reared in ponds (800 m3 volume) to determine the effects of tadpole density and carbaryl exposure on mass at metamorphosis and on time and survival to metamorphosis. Exposure to carbaryl significantly affected toads at metamorphosis, but not leopard frogs. Carbaryl exposure nearly doubled toad survival compared to controls; this effect may be attributable to an indirect effect of carbaryl increasing algal food resources. The competitive environment (i.e., density) and carbaryl exposure significantly affected the trade-off between mass and time to metamorphosis for toads. Our study is the first to demonstrate that in pond communities where predation and competition may be strong, short-lived insecticides can significantly alter the community dynamics of amphibians.

Because declines within amphibian populations can seldom be attributed to a single cause, it is important to focus on multiple stressors, both natural and anthropogenic. Variables such as UV-B radiation and chemical contamination can interact with one another in ways that might not be predicted from single-factor studies. We exposed southern leopard frog (Rana sphenocephala) tadpoles to the insecticide carbaryl and varying intensities of UV-B radiation in artificial ponds and examined their effects on survival, size at metamorphosis, and the duration of the larval period. Tadpole survival to metamorphosis was positively influenced by UV-B intensity. Tadpoles in ponds exposed to carbaryl contained over three times more algae and yielded larger metamorphs than control ponds. Although previous laboratory studies have indicated carbaryl becomes more toxic in the presence of UV-B, we did not find such an effect, perhaps because of the protection afforded by dissolved organic carbon within the ponds. Our research emphasizes the importance of conducting field studies to more accurately predict what occurs under a natural setting.