Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants of growing concern due to their potential negative effects on wildlife and human health. Per- and polyfluoroalkyl substances have been shown to alter immune function in various taxa, which could influence the outcomes of host-parasite interactions. To date, studies have focused on the effects of PFAS on host susceptibility to parasites, but no studies have addressed the effects of PFAS on parasites. To address this knowledge gap, we independently manipulated exposure of larval northern leopard frogs (Rana pipiens) and parasites (flatworms) via their snail intermediate host to environmentally relevant PFAS concentrations and then conducted trials to assess host susceptibility to infection, parasite infectivity, and parasite longevity after emergence from the host. We found that PFAS exposure to only the host led to no significant change in parasite load, whereas exposure of parasites to a 10-µg/L mixture of PFAS led to a significant reduction in parasite load in hosts that were not exposed to PFAS. We found that when both host and parasite were exposed to PFAS there was no difference in parasite load. In addition, we found significant differences in parasite longevity post emergence following exposure to PFAS. Although some PFAS-exposed parasites had greater longevity, this did not necessarily translate into increased infection success, possibly because of impaired movement of the parasite. Our results indicate that exposure to PFAS can potentially impact host-parasite interactions. Environ Toxicol Chem 2024;43:1537-1546. © 2024 SETAC.

Short-chain perfluoroalkyl carboxylic acids (PFCAs) have been detected in the environment globally. The presence and persistence of these compounds in the environment may lead to chronic wildlife exposure. We used northern leopard frog (Rana pipiens) tadpoles to investigate the chronic toxicity and the bioconcentration of two short-chain PFCAs, perfluorobutanoic acid (PFBA) and perfluorohexanoic acid (PFHxA). We exposed Gosner stage 25 tadpoles to PFBA and PFHxA (as individual chemicals) at nominal concentrations of 0.1, 1, 10, 100, and 1000 µg/L for 43-46 days. Tadpoles exposed to 0.1 to 100 µg/L of PFBA and PFHxA had significantly higher mean snout-to-vent lengths, mean masses, and scaled mass indexes than control tadpoles. These results indicate that exposure to short-chain PFCAs influences tadpole growth. Further investigation into the mechanism(s) causing the observed changes in tadpole growth is warranted. We observed a significantly higher proportion of males in the PFBA 1 µg/L treatment group, however further histological analyses are required to confirm visual sex identification before making concrete conclusions on the effects of PFCAs on amphibian sex ratios. PFBA concentrations in tissues were higher than PFHxA concentrations; a pattern that contrasts with previously published studies using fish, suggesting potential differences between taxa in PFBA and PFHxA bioconcentration. Bioconcentration factors were <10 L/kg wet weight, indicating low bioconcentration potential in tadpoles. Our results suggest that PFBA and PFHxA may have effects at environmentally-relevant concentrations (0.1-10 µg/L) and further investigation is required before these compounds can be deemed a \"safe\" alternative to their long-chain counterparts.

Per- and polyfluoroalkyl substances (PFAS) occur in the environment as mixtures, yet mixture toxicity remains poorly understood. Aqueous film-forming foams (AFFFs) are a common source of PFAS. Our objective was to examine chronic effects of a complex PFAS mixture on amphibian growth and development. We tested toxicity of a five-chemical PFAS mixture summing to 10 μg/L and that accounts for >90% of the PFAS in AFFF-affected surface waters: perfluorooctane sulfonate (PFOS, 40%), perfluorohexane sulfonic acid (PFHxS, 30%), perflurooctanoic acid (PFOA, 12.5%), perfluorohexanoic acid (PFHxA, 12.5%), and perfluoropentanoic acid (PFPeA, 5%). We also included treatments to determine whether PFOS drove mixture toxicity and whether PFOS and mixture components act additively. We exposed Northern leopard frog (Rana pipiens) larvae through metamorphosis (∼130 d) in outdoor mesocosms. After 21 days of exposure, the larval body condition fell ∼5% relative to controls in the 4 μg/L PFOS treatment and mixtures lacking PFOS. At metamorphosis, the full 5-component 10 μg/L PFAS mixture reduced mass by 16% relative to controls. We did not observe effects on development. Our results indicate that toxicity of PFOS and other PFAS mixtures typical of AFFF sites act additively and that PFOS is not more inherently toxic than other mixture components.

This prospective, descriptive study focused on lung flukes (Hematoloechus sp., H) and their impact on systemic and individual capillary variables measured in pithed Rana pipiens, a long-standing model for studies of capillary physiology. Three groups were identified based on Hematoloechus attachment: no Hematoloechus (No H), Hematoloechus not attached (H Not Att), and Hematoloechus attached (H Att). Among 38 descriptive, cardiovascular, and immunological variables, 18 changed significantly with H. Symptoms of H included weight loss, elevated immune cells, heart rate variability, faster coagulation, lower hematocrit, and fluid accumulation. Important capillary function discoveries included median baselines for hydraulic conductivity (Lp) of 7.0 (No H), 12.4 (H Not Att), and 4.2 (H Att) × 10-7 cm·s-1·cmH2O-1 (P < 0.0001) plus seasonal adaptation of sigma delta pi [σ(πc-πi), P = 0.03]. Pro- and anti-inflammatory phases were revealed for Lp and plasma nitrite/nitrate concentration ([NOx]) in both H Not Att and H Att, whereas capillary wall tensile strength increased in the H Att. H attachment was advantageous for the host due to lower edema and for the parasite via a sustained food source illustrating an excellent example of natural symbiosis. However, H attachment also resulted in host weight loss: in time, a conundrum for the highly dependent parasite. The study increases overall knowledge of Rana pipiens by revealing intriguing effects of H and previously unknown, naturally occurring seasonal changes in many variables. The data improve Rana pipiens as a general scientific and capillary physiology model. Diseases of inflammation and stroke are among the clinical applications.

Neonicotinoids are neurotoxic insecticides and are often released into nearby wetlands via subsurface tile drains and can negatively impact nontarget organisms, such as amphibians. Previous studies have indicated that imidacloprid, a commonly used neonicotinoid, can cross the amphibian blood-brain barrier under laboratory conditions; however, little is known about the impact of low concentrations in a field-based setting. Here, we report aqueous pesticide concentrations at wetland production areas that were either connected or not connected to agricultural tile drains, quantified imidacloprid and its break down products in juvenile amphibian brains and livers, and investigated the relationship between imidacloprid brain concentration and brain size. Imidacloprid concentrations in brain and water samples were nearly 2.5 and 5 times higher at tile wetlands (brain = 4.12 ± 1.92 pg/mg protein; water = 0.032 ± 0.045 μg/L) compared to reference wetlands, respectively. Tile wetland amphibians also had shorter cerebellums (0.013 ± 0.001 mm), depicting a negative relationship between imidacloprid brain concentration and cerebellum length. The metabolite, desnitro-imidacloprid, had liver concentrations that were 2 times higher at tile wetlands (2 ± 0.3 μg/g). Our results demonstrate that imidacloprid can cross the amphibian blood-brain barrier under ecological conditions and may alter brain dimensions and provide insight into the metabolism of imidacloprid in amphibians.

Pond management with chemical and biological agents that reduce overgrowth of algae is an important means of maintaining water quality in residential ponds, yet the effects on nontarget species are not fully understood. We assessed the impact of Aquashade (a common nontoxic pond dye) and copper sulfate (a toxic algaecide) on American toad (Anaxyrus americanus), northern leopard frog (Lithobates pipiens), and Cope\'s gray treefrog (Hyla chrysoscelis) metamorphosis in outdoor mesocosm experiments. We also evaluated the relative impact of tadpole grazing versus chemical treatment on phytoplankton and periphyton abundance. We found no significant effects of pond management treatment on anuran metamorphosis, suggesting that addition of Aquashade and copper sulfate at tested concentrations does not significantly impact anurans under these experimental conditions. Interestingly, we found that the presence of tadpoles more strongly reduced algal abundance than Aquashade or copper sulfate by significantly decreasing phytoplankton and periphyton abundance over time. The present study suggests that anuran tadpoles may be effective at maintaining water quality, and that Aquashade and copper sulfate may have minimal effects on amphibian metamorphosis. Environ Toxicol Chem 2023;42:213-224. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Per- and polyfluoroalkyl substances (PFAS) are chemicals associated with adverse health effects. At aqueous film-forming foam sites, they occur as mixtures, with perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) commonly co-occurring in the highest concentrations. Although PFOS and PFHxS toxicities have been studied, few studies have tested their potential interaction. Using Rana pipiens, the present study compared toxicities of a 1:1 PFOS:PFHxS mixture to PFOS and PFHxS individually with the prediction that responses would be additive. Gosner stage 25 (GS 25) tadpoles were exposed through metamorphosis (GS 46) to 0.5 and 1 ppb PFOS or PFHxS alone or to a mixture of 0.5 ppb PFOS and 0.5 ppb PFHxS. Tadpoles were weighed and measured (snout-vent length [SVL]) at day 31, metamorphic climax (GS 42), and GS 46. These values were used to calculate the scaled mass index (SMI), a measure of body condition. Body burdens were quantified on day 31 and at GS 46. The PFOS and PFHxS body burdens were elevated relative to controls at GS 46. No effects were observed on survival, SVL, or mass. Single PFAS effects included a 17% reduction in SMI at day 31 (0.5 ppb PFHxS) and a 1.1-day longer metamorphic period (1 ppb PFHxS) relative to controls. Mixture results deviated from additivity-SMIs were higher than expected on day 31 and lower than expected at GS 42. In addition, time to GS 42 in the PFAS mixture exceeded expected additivity by 12 days. Results from a chronic exposure to a 1:1 PFOS:PFHxS mixture resulted in changes in body condition and length of metamorphosis that deviated from additivity. More PFAS mixture toxicity studies conducted at relevant ratios and concentrations are needed. Environ Toxicol Chem 2022;41:3007-3016. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Differences in morphology, ecology, and behavior through ontogeny can result in opposing selective pressures at different life stages. Most animals, however, transition through two or more distinct phenotypic phases, which is hypothesized to allow each life stage to adapt more freely to its ecological niche. How this applies to sensory systems, and in particular how sensory systems adapt across life stages at the molecular level, is not well understood. Here, we used whole-eye transcriptomes to investigate differences in gene expression between tadpole and juvenile southern leopard frogs (Lithobates sphenocephalus), which rely on vision in aquatic and terrestrial light environments, respectively. Because visual physiology changes with light levels, we also tested the effect of light and dark exposure.
We found 42% of genes were differentially expressed in the eyes of tadpoles versus juveniles and 5% for light/dark exposure. Analyses targeting a curated subset of visual genes revealed significant differential expression of genes that control aspects of visual function and development, including spectral sensitivity and lens composition. Finally, microspectrophotometry of photoreceptors confirmed shifts in spectral sensitivity predicted by the expression results, consistent with adaptation to distinct light environments.
Overall, we identified extensive expression-level differences in the eyes of tadpoles and juveniles related to observed morphological and physiological changes through metamorphosis and corresponding adaptive shifts to improve vision in the distinct aquatic and terrestrial light environments these frogs inhabit during their life cycle. More broadly, these results suggest that decoupling of gene expression can mediate the opposing selection pressures experienced by organisms with complex life cycles that inhabit different environmental conditions throughout ontogeny.

Host species that can independently maintain a pathogen in a host community and contribute to infection in other species are important targets for disease management. However, the potential of host species to maintain a pathogen is not fixed over time, and an important challenge is understanding how within- and across-season variability in host maintenance potential affects pathogen persistence over longer time scales relevant for disease management (e.g., years). Here, we sought to understand the causes and consequences of seasonal infection dynamics in leopard frogs (Rana sphenocephala and Rana pipiens) infected with the fungal pathogen Batrachochytrium dendrobatidis (Bd). We addressed three questions broadly applicable to seasonal host-parasite systems. First, to what degree are observed seasonal patterns in infection driven by temperature-dependent infection processes compared to seasonal host demographic processes? Second, how does seasonal variation in maintenance potential affect long-term pathogen persistence in multi-host communities? Third, does high deterministic maintenance potential relate to the long-term stochastic persistence of pathogens in host populations with seasonal infection dynamics? To answer these questions, we used field data collected over 3 years on >1400 amphibians across four geographic locations, laboratory and mesocosm experiments, and a novel mathematical model. We found that the mechanisms that drive seasonal prevalence were different from those driving seasonal infection intensity. Seasonal variation in Bd prevalence was driven primarily by changes in host contact rates associated with breeding migrations to and from aquatic habitat. In contrast, seasonal changes in infection intensity were driven by temperature-induced changes in Bd growth rate. Using our model, we found that the maintenance potential of leopard frogs varied significantly throughout the year and that seasonal troughs in infection prevalence made it unlikely that leopard frogs were responsible for long-term Bd persistence in these seasonal amphibian communities, highlighting the importance of alternative pathogen reservoirs for Bd persistence. Our results have broad implications for management in seasonal host-pathogen systems, showing that seasonal changes in host and pathogen vital rates, rather than the depletion of susceptible hosts, can lead to troughs in pathogen prevalence and stochastic pathogen extirpation.

Although there is mounting evidence indicating that the relative timing of predator and prey phenologies determines the outcome of trophic interactions, we still lack a comprehensive understanding of how the environmental context (e.g., abiotic conditions) influences this relationship. Environmental conditions not only frequently drive shifts in phenologies, but they can also affect the very same processes that mediate the effects of phenological shifts on species interactions. Therefore, identifying how environmental conditions shape the effects of phenological shifts is key to predicting community dynamics across a heterogeneous landscape and how they will change with ongoing climate change in the future. Here I tested how environmental conditions shape the effects of phenological shifts by experimentally manipulating temperature, nutrient availability, and relative phenologies in two predator-prey freshwater systems (mole salamander-bronze frog vs. dragonfly larvae-leopard frog). This allowed me to (1) isolate the effects of phenological shifts and different environmental conditions; (2) determine how they interact; and (3) evaluate how consistent these patterns are across different species and environments. I found that delaying prey arrival dramatically increased predation rates, but these effects were contingent on environmental conditions and the predator system. Although nutrient addition and warming both significantly enhanced the effect of arrival time, their effect was qualitatively different across systems: Nutrient addition enhanced the positive effect of early arrival in the dragonfly-leopard frog system, whereas warming enhanced the negative effect of arriving late in the salamander-bronze frog system. Predator responses varied qualitatively across predator-prey systems. Only in the system with a strong gape limitation were predators (salamanders) significantly affected by prey arrival time and this effect varied with environmental context. Correlations between predator and prey demographic rates suggest that this was driven by shifts in initial predator-prey size ratios and a positive feedback between size-specific predation rates and predator growth rates. These results highlight the importance of accounting for temporal and spatial correlations of local environmental conditions and gape limitation when predicting the effects of phenological shifts and climate change on predator-prey systems.