Effects of perfluoroalkyl sulfonic acids on developmental, physiological, and immunological measures in northern leopard frog tadpoles.

The chronic toxicity of short chain perfluoroalkyl sulfonic acids (PFSAs), such as perfluorobutanesulfonic acid (PFBS) and perfluorohexanesulfonic acid (PFHxS), are relatively understudied despite the increasing detection of these compounds in the environment. We investigated the chronic toxicity and bioconcentration of PFBS and PFHxS using northern leopard frog (Rana [Lithobates] pipiens) tadpoles. We exposed Gosner stage (GS) 25 tadpoles to either PFBS or PFHxS at nominal concentrations of 0.1, 1, 10, 100, and 1000 µg/L until metamorphosis (GS42). We then assessed tadpole growth, development, stress, and immune metrics, and measured fatty acid (FA) composition and PFSA concentrations in liver and whole-body tissues. Tadpole growth and development measures were relatively unaffected by PFSA exposure. However, tadpoles exposed to 1000 µg/L PFBS or PFHxS had significantly increased hepatosomatic indexes (HSI) relative to controls. Further, tadpoles from the 1000 µg/L PFHxS treatment had altered FA profiles relative to controls, with increased total FAs, saturated FAs, monounsaturated FAs, and omega-6 polyunsaturated FAs. In addition, tadpoles from the 1000 µg/L PFHxS treatment had a higher probability of waterborne corticosterone detection. These results suggest that PFBS and PFHxS influence the hepatic health of tadpoles, and that PFHxS may alter lipid metabolism in tadpoles. We also observed a higher probability of tadpoles being phenotypically female after exposure to an environmentally relevant concentration (0.1 µg/L) of PFHxS, suggesting that PFHxS may exert endocrine disrupting effects on tadpoles during early development. The measured bioconcentration factors (BCFs) for both compounds were ≤10 L kg-1 wet weight, suggesting low bioconcentration potential for PFBS and PFHxS in tadpoles. Many of the significant effects observed in this study occurred at concentrations several orders of magnitude above those measured in the environment; however, our work shows effects of PFSAs exposure on amphibians and provides essential information for ecological risk assessments of these compounds.

Diverse Relationships between Batrachochytrium Infections and Antimicrobial Peptide Defenses Across Leopard Frog Populations.

Antimicrobial peptides (AMPs) play a fundamental role in the innate defense against microbial pathogens, as well as other immune and non-immune functions. Their role in amphibian skin defense against the pathogenic fungus Batrachochytrium dendrobatidis (Bd) is exemplified by experiments in which depletion of host's stored AMPs increases mortality from infection. Yet, the question remains whether there are generalizable patterns of negative or positive correlations between stored AMP defenses and the probability of infection or infection intensity across populations and species. This study aims to expand on prior field studies of AMP quantities and compositions by correlating stored defenses with an estimated risk of Bd exposure (prevalence and mean infection intensity in each survey) in five locations across the United States and a total of three species. In all locations, known AMPs correlated with the ability of recovered secretions to inhibit Bd in vitro. We found that stored AMP defenses were generally unrelated to Bd infection except in one location where the relative intensity of known AMPs was lower in secretions from infected frogs. In all other locations, known AMP relative intensities were higher in infected frogs. Stored peptide quantity was either positively or negatively correlated with Bd exposure risk. Thus, future experiments coupled with organismal modeling can elucidate whether Bd infection affects secretion/synthesis and will provide insight into how to interpret amphibian ecoimmunology studies of AMPs. We also demonstrate that future AMP isolating and sequencing studies can focus efforts by correlating mass spectrometry peaks to inhibitory capacity using linear decomposition modeling.

Assessing the Combined Effects of Host and Parasite Exposure to Forever Chemicals in an Amphibian-Echinostome System.

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.

The effects of two short-chain perfluoroalkyl carboxylic acids (PFCAs) on northern leopard frog (Rana pipiens) tadpole development.

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.

Chronic Exposure to a PFAS Mixture Resembling AFFF-Impacted Surface Water Decreases Body Size in Northern Leopard Frogs (Rana pipiens).

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.

Hematoloechus sp. attachment shifts endothelium in vivo from pro- to anti-inflammatory profile in Rana pipiens: evidence from systemic and capillary physiology.

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.

Connecting the Pipes: Agricultural Tile Drains and Elevated Imidacloprid Brain Concentrations in Juvenile Northern Leopard Frogs (Rana pipiens).

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.

Assessing the Impact of Chemical Algae Management Strategies on Anurans and Aquatic Communities.

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.

An Environmentally Relevant Mixture of Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Does Not Conform to Additivity in Northern Leopard Frogs Exposed Through Metamorphosis.

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.

Transcriptomic evidence for visual adaptation during the aquatic to terrestrial metamorphosis in leopard frogs.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Temperature and nutrient conditions modify the effects of phenological shifts in predator-prey communities.

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.

Comparative Toxicity of Aquatic Per- and Polyfluoroalkyl Substance Exposure in Three Species of Amphibians.

Per- and polyfluoroalkyl substances (PFAS) are contaminants of concern due to their widespread occurrence in the environment, persistence, and potential to elicit a range of negative health effects. Per- and polyfluoroalkyl substances are regularly detected in surface waters, but their effects on many aquatic organisms are still poorly understood. Species with thyroid-dependent development, like amphibians, can be especially susceptible to PFAS effects on thyroid hormone regulation. We examined sublethal effects of aquatic exposure to four commonly detected PFAS on larval northern leopard frogs (Rana [Lithobates] pipiens), American toads (Anaxyrus americanus), and eastern tiger salamanders (Ambystoma tigrinum). Animals were exposed for 30 days (frogs and salamanders) or until metamorphosis (toads) to 10, 100, or 1000 µg/L of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), or 6:2 fluorotelomer sulfonate (6:2 FTS). We determined that chronic exposure to common PFAS can negatively affect amphibian body condition and development at concentrations as low as 10 µg/L. These effects were highly species dependent, with species having prolonged larval development (frogs and salamanders) being more sensitive to PFAS than more rapidly developing species (toads). Our results demonstrate that some species could experience sublethal effects at sites with surface waters highly affected by PFAS. Our results also indicate that evaluating PFAS toxicity using a single species may not be sufficient for accurate amphibian risk assessment. Future studies are needed to determine whether these differences in susceptibility can be predicted from species' life histories and whether more commonly occurring environmental levels of PFAS could affect amphibians. Environ Toxicol Chem 2022;41:1407-1415. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Detection of imidacloprid and metabolites in Northern Leopard frog (Rana pipiens) brains.

Neonicotinoids are a new type of highly water-soluble insecticide used in agricultural practices to eliminate pests. Neonicotinoids bind almost irreversibly to postsynaptic nicotinic acetylcholine receptors in the central nervous system of invertebrates, resulting in overstimulation, paralysis, and death. Imidacloprid, the most commonly used neonicotinoid, is often transported to nearby wetlands through subsurface tile drains and has been identified as a neurotoxin in several aquatic non-target organisms. The aim of the present study was to determine if imidacloprid could cross the blood-brain barrier in adult Northern Leopard frogs (Rana pipiens) following exposure to 0, 0.1, 1, 5, or 10 µg/L for 21 days. Additionally, we quantified the breakdown product of imidacloprid, imidacloprid-olefin, and conducted feeding trials to better understand how imidacloprid affects foraging behavior over time. Exposure groups had 12 to 313 times more imidacloprid in the brain relative to the control and breakdown products showed a dose-response relationship. Moreover, imidacloprid brain concentrations were approximately 14 times higher in the 10 µg/L treatment compared to the water exposure concentration, indicating imidacloprid can bioaccumulate in the amphibian brain. Reaction times to a food stimulus were 1.5 to 3.2 times slower among treatment groups compared to the control. Furthermore, there was a positive relationship between mean response time and log-transformed imidacloprid brain concentration. These results indicate imidacloprid can successfully cross the blood-brain barrier and bioaccumulate in adult amphibians. Our results also provide insights into the relationship between imidacloprid brain concentration and subsequent altered foraging behavior.

Induced Hepatic Glutathione and Metabolomic Alterations Following Mixed Pesticide and Fertilizer Exposures in Juvenile Leopard Frogs (Lithobates sphenocephala).

The increasing use of agrochemicals, alone and in combination, has been implicated as a potential causative factor in the decline of amphibians worldwide. Fertilizers and pesticides are frequently combined into single-use tank mixtures for agricultural applications to decrease costs while meeting the food demands of a growing human population. Limited data are available on the effects of increased nitrogen levels in nontarget species, such as amphibians, and therefore investigating alterations in the nitrogen cycle and its impacts on amphibians needs to be considered in best management practices going forward. The objective of the present study was to elucidate the impact of fertilizer (urea) and herbicide (atrazine and/or alachlor) tank mixtures on the hepatic metabolome of juvenile leopard frogs as well as to investigate alterations in oxidative stress by relating these changes to glutathione (GSH) levels. Herbicide exposure only moderately increased this parameter in amphibians, however, urea alone and in combination with either atrazine or alachlor statistically elevated GSH levels. Interestingly, urea also inhibited pesticide uptake: calculated bioconcentration factors were greatly decreased for atrazine and alachlor when urea was present in the exposure mixture. Metabolomic profiling identified fluxes in hepatic metabolites that are involved in GSH and carbohydrate metabolic processes as well as altered intermediates in the urea cycle. Ultimately, understanding the biological impacts of nitrogenous fertilizers alone and in combination with pesticide exposure will inform best management practices to conserve declining amphibian populations worldwide. Environ Toxicol Chem 2022;41:122-133. © 2021 SETAC.

Effects of salinity and a glucocorticoid antagonist, RU486, on waterborne aldosterone and corticosterone of northern leopard frog larvae.

Increased salinity is an emerging contaminant of concern for aquatic taxa. For amphibians exposed to salinity, there is scarce information about the physiological effects and changes in osmoregulatory hormones such as corticosterone (CORT) and aldosterone (ALDO). Recent studies have quantified effects of salinity on CORT physiology of amphibians based on waterborne hormone collection methods, but much less is known about ALDO in iono- and osmoregulation of amphibians. We re-assayed waterborne hormone samples from a previous study to investigate effects of salinity (sodium chloride, NaCl) and a glucocorticoid receptor antagonist (RU486) on ALDO of northern leopard frog (Rana pipiens) larvae. We also investigated relationships between ALDO and CORT. Waterborne ALDO marginally decreased with increasing salinity and was, unexpectedly, positively correlated with baseline and stress-induced waterborne CORT. Importantly, ALDO increased when larvae were exposed to RU486, suggesting that RU486 may also suppress mineralocorticoid receptors or that negative feedback of ALDO is mediated through glucocorticoid receptors. Alternatively, CORT increases with RU486 treatment and might be a substrate for ALDO synthesis, which could account for increases in ALDO with RU486 treatment and the correlation between CORT and ALDO. ALDO was negatively correlated with percent water, such that larvae secreting more ALDO retained less water. Although sample sizes were limited and further validation and studies are warranted, our findings expand our understanding of adrenal steroid responses to salinization in amphibians and proposes new hypotheses regarding the co-regulation of ALDO and CORT.

Larval Exposure to Polychlorinated Biphenyl-126 Led to a Long-Lasting Decrease in Immune Function in Postmetamorphic Juvenile Northern Leopard Frogs, Lithobates pipiens.

Amphibian populations are decreasing worldwide, and pollution is a contributing factor. Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants known to exert immunotoxicity. To assess impacts of PCBs on frogs, we exposed Lithobates pipiens tadpoles to a diet of PCB-126 (0-5 ng PCB-126/g wet food) through metamorphic climax. Postmetamorphic frogs were immunized with keyhole limpet hemocyanin (KLH); then production of KLH-specific IgY, as well as total IgY and IgM, was measured (Trial I). A second larval study (0 and 7.3 ng PCB-126/g wet food) was performed to investigate whether PCB altered antigenic responses in prometamorphic tadpoles (Gosner Stage 36-39), and to measure the innate immune response of postmetamorphic frogs (Trial II). After larval PCB-126 exposure, both KLH-specific IgY levels and complement activity were reduced. Because postmetamorphic frogs carried a body burden of PCB-126 (2.4 ng/g or less), we wanted to determine whether the effect on immune response was due to larval exposure or to the resulting body burden as frogs. To test this, we reared tadpoles under control conditions (no PCB), and limited PCB exposure to postmetamorphosis only by injecting 2-week-old frogs with 10 ng PCB-126/g (Trial III). The resulting body burden (3.4 ng/g) was similar to that of frogs in Trial I, but we no longer detected suppression of KLH-specific IgY or hemolytic activity. These results suggest life-stage-specific immune responses; however, because we administered PCB-126 differently between trials, it is premature to conclude that these differences are intrinsically life stage dependent, and further study is warranted. Regardless, our study demonstrated a long-lasting effect of larval PCB-126 exposure that persisted through metamorphosis and suppressed frog immunity. Environ Toxicol Chem 2022;41:81-94. © 2021 SETAC.

Retinoic acid treatment recruits macrophages and increases axonal regeneration after optic nerve injury in the frog Rana pipiens.

Retinoic acid (RA) plays major roles during nervous system development, and during regeneration of the adult nervous system. We have previously shown that components of the RA signaling pathway are upregulated after optic nerve injury, and that exogenous application of all-trans retinoic acid (ATRA) greatly increases the survival of axotomized retinal ganglion cells (RGCs). The objective of the present study is to investigate the effects of ATRA application on the macrophages in the optic nerve after injury, and to determine whether this affects axonal regeneration. The optic nerve was crushed and treated with PBS, ATRA and/or clodronate-loaded liposomes. Nerves were examined at one and two weeks after axotomy with light microscopy, immunocytochemistry and electron microscopy. ATRA application to the optic nerve caused transient increases in the number of macrophages and microglia one week after injury. The macrophages are consistently labeled with M2-type markers, and have considerable phagocytic activity. ATRA increased ultrastructural features of ongoing phagocytic activity in macrophages at one and two weeks. ATRA treatment also significantly increased the numbers of regenerating GAP-43-labeled axons. Clodronate liposome treatment depleted macrophage numbers by 80%, completely eliminated the ATRA-mediated increase in axonal regeneration, and clodronate treatment alone decreased axonal numbers by 30%. These results suggest that the success of axon regeneration is partially dependent on the presence of debris-phagocytosing macrophages, and that the increases in regeneration caused by ATRA are in part due to their increased numbers. Further studies will examine whether macrophage depletion affects RGC survival.

Comparison of Thermal and Mechanical Noxious Stimuli for Testing Analgesics in White's Tree Frogs (Litoria caerulea) and Northern Leopard Frogs (Lithobates pipiens).

Determining the clinical efficacy of analgesic drugs in amphibians can be particularly challenging. The current study investigated whether a thermal nociceptive stimulus is useful for the evaluation of analgesic drugs in 2 amphibian species. The objectives of this study were 2-fold: 1) compare 2 models of nociception (thermal and mechanical) using 2 frog species; White's Tree Frogs (Litoria caerulea; WTF) and Northern Leopard Frogs (Lithobates pipiens; NLF) after administration of saline or morphine sulfate; and 2) evaluate antinociceptive efficacy of morphine sulfate at 2 doses in a common amphibian research species, the NLF, using a mechanical stimulus. Neither WTF nor NLF displayed consistent drug-dependent changes in withdrawal responses to a noxious thermal stimulus applied using the Hargreaves apparatus, but NLF exposed to the noxious mechanical stimulus demonstrated a significant dose-dependent antinociceptive response to morphine sulfate. These results indicate that morphine is not antinociceptive in WTF, supporting previously reported results, and demonstrate the importance of using an appropriate experimental antinociceptive test in amphibians. Our data suggest that nociception in amphibian species may be best evaluated by using mechanical nociceptive models, although species differences must also be considered.

The Effects of Dietary Polybrominated Diphenyl Ether Exposure and Rearing Temperature on Tadpole Growth, Development, and Their Underlying Processes.

Depression of growth rate due to polybrominated diphenyl ethers (PBDEs) has been documented in birds, mammals, amphibians, and fish at single temperatures. However, the underlying energetic mechanism for this effect and how it might change in relation to changing environmental temperature remain unstudied. We used a simple energy budget to address hypotheses regarding effects of PBDEs on tadpole (Lithobates pipiens) growth: that reductions in growth are linked to increased respiratory costs, reductions in digestive performance, differences in body composition, reductions in food intake, or a combination of these factors. From 18 days postfertilization (dpf) until 42 dpf, tadpoles were exposed dietarily to a pentabromodiphenyl ether mixture (DE-71TM ) at a concentration of 100 ng DE-71/g wet mass under a rearing temperature of either 22 or 27 °C. After 20 days of PBDE exposure, total PBDEs in tadpoles averaged 148.4 ng/g wet mass, with no differences by rearing temperature and approximately 50% higher than in their diet; controls not fed PBDE had levels <1 ng/g. Exposure to PBDE resulted in reductions in body length, mass, and development compared to controls, independent of rearing temperature; PBDE had no effect on measures of body composition, dry matter digestibility, or oxygen consumption. A simple energy budget using data from the present study revealed that a 10% decrease in feeding rate could explain the lower mass gain of tadpoles exposed to PBDE. Growth depression by PBDE could be due to (1) direct inhibition of growth processes by PBDE that indirectly decreases total energy demand and food intake, and (2) direct inhibition of food intake. Future studies to disentangle these possible pathways of PBDE effects are warranted. Environ Toxicol Chem 2021;40:3181-3192. © 2021 SETAC.

Comparative Effects of Energy-Related Saline Wastewaters and Sodium Chloride on Hatching, Survival, and Fitness-Associated Traits of Two Amphibian Species.

Salinity (sodium chloride [NaCl]) is a prevalent and persistent contaminant that negatively affects freshwater ecosystems. Although most studies focus on effects of salinity from road salts (primarily NaCl), high-salinity wastewaters from energy extraction (wastewaters) could be more harmful because they contain NaCl and other toxic components. Many amphibians are sensitive to salinity, and their eggs are thought to be the most sensitive life-history stage. However, there are few investigations with salinity that include eggs and larvae sequentially in long-term exposures. We investigated the relative effects of wastewaters from a large energy reserve, the Williston Basin (USA), and NaCl on northern leopard (Rana pipiens) and boreal chorus (Pseudacris maculata) frogs. We exposed eggs and tracked responses through larval stages (for 24 days). Wastewaters and NaCl caused similar reductions in hatching and larval survival, growth, development, and activity, while also increasing deformities. Chorus frog eggs and larvae were more sensitive to salinity than leopard frogs, suggesting species-specific responses. Contrary to previous studies, eggs of both species were less sensitive to salinity than larvae. Our ecologically relevant exposures suggest that accumulating effects can reduce survival relative to starting experiments with unexposed larvae. Alternatively, egg casings of some species may provide some protection against salinity. Notably, effects of wastewaters on amphibians were predominantly due to NaCl rather than other components. Therefore, findings from studies with other sources of increased salinity (e.g., road salts) could guide management of wastewater-contaminated ecosystems, and vice versa, to mitigate effects of salinization. Environ Toxicol Chem 2021;40:3137-3147. © 2021 SETAC.