Warmer temperature increases toxicokinetic elimination of PCBs and PBDEs in Northern leopard frog larvae (Lithobates pipiens).

We studied the temperature dependence of accumulation and elimination of two polychlorinated biphenyls (PCBs; PCB-70 and PCB-126) and a commercial mixture of congeners of polybrominated diphenyl ethers (PBDEs; DE-71™)) in Northern leopard frog (Lithobates pipiens) tadpoles. We reared tadpoles at 18, 23, or 27 °C for 5.3 or up to 13.6 weeks (longer at cooler temperature where development is slower) on diets containing the toxicants, each at several different toxicant concentrations, and compared tissue concentrations as a function of food concentration and rearing temperature. Following > 1 month of accumulation, tissue concentrations of all three toxicants in exposed tadpoles were linearly related to dietary concentrations as expected for first order kinetics, with no significant effect of rearing temperature.We also raised free-swimming L. pipiens tadpoles for 14 days on foods containing either toxicant at 18 or 27 °C during an accumulation phase, and then during depuration (declining toxicant) phase of 14 days we provided food without toxicants and measured the decline of toxicants in tadpole tissue. All the congeners were eliminated faster at warmer rearing temperature, as expected. Using Arrhenius' equation, we calculated that the apparent activation energy for elimination of both PCB congeners by tadpoles was 1.21 eV (95% confidence interval 0.6-1.8 eV). We discuss how this value was within the range of estimates for metabolic reactions generally (range 0.2 - 1.2 eV), which might include metabolic pathways for biotransformation and elimination of PCBs. Furthermore, we discuss how the lack of an effect of rearing temperature on tadpole near-steady-state tissue residue levels suggests that faster elimination at the warmer temperature was balanced by faster uptake, which is plausible considering the similar temperature sensitivities (i.e., activation energies) of all these processes. Although interactions between toxicants and temperature can be complex and likely toxicant-dependent, it is plausible that patterns observed in tadpoles might apply to other aquatic organisms. Published data on depuration in 11 fish species eliminating 8 other organic toxicants indicated that they also had similar apparent activation energy for elimination (0.82 ± 0.12 eV; 95% confidence interval 0.56 - 1.08 eV), even though none of those studied toxicants were PCBs or PBDEs. Additional research on toxicant-temperature interactions can help improve our ability to predict toxicant bioaccumulation in warming climate scenarios.

Timing and order of exposure to two echinostome species affect patterns of infection in larval amphibians.

The study of priority effects with respect to coinfections is still in its infancy. Moreover, existing coinfection studies typically focus on infection outcomes associated with exposure to distinct sets of parasite species, despite that functionally and morphologically similar parasite species commonly coexist in nature. Therefore, it is important to understand how interactions between similar parasites influence infection outcomes. Surveys at seven ponds in northwest Pennsylvania found that multiple species of echinostomes commonly co-occur. Using a larval anuran host (Rana pipiens) and the two most commonly identified echinostome species from our field surveys (Echinostoma trivolvis and Echinoparyphium lineage 3), we examined how species composition and timing of exposure affect patterns of infection. When tadpoles were exposed to both parasites simultaneously, infection loads were higher than when exposed to Echinoparyphium alone but similar to being exposed to Echinostoma alone. When tadpoles were sequentially exposed to the parasite species, tadpoles first exposed to Echinoparyphium had 23% lower infection loads than tadpoles first exposed to Echinostoma. These findings demonstrate that exposure timing and order, even with similar parasites, can influence coinfection outcomes, and emphasize the importance of using molecular methods to identify parasites for ecological studies.

A Bacillus thuringiensis kurstaki Biopesticide Does Not Reduce Hatching Success or Tadpole Survival at Environmentally Relevant Concentrations in Southern Leopard Frogs (Lithobates sphenocephalus).

Amphibians are in global decline, and anthropogenic activities are known leading causes of their demise. Thus the interaction between agriculture and amphibian health has been examined for decades. Many facets of amphibian physiology and ecology place them at high risk among the nontarget organisms affected by agricultural byproducts. Research has shown that many chemicals and fertilizers affect amphibian growth, reproduction, and survival. The impacts differ based on the type of agricultural byproduct (e.g., chemical pesticide or nutrient-heavy fertilizer) and amphibian species, but the effects are usually negative. However, minimal research exists on how organic biopesticides interact with amphibian populations. Biopesticides utilize insecticidal bacteria as the active ingredient in lieu of synthetic chemicals. The inert ingredients present in biopesticide commercial products are considered safe to nontarget organisms. The present study tested the impacts of a commercial biopesticide on the survival of amphibian embryos and larvae. We found that expected environmental concentrations of the microbial biopesticide Monterrey B.t. did not significantly reduce survival in embryos or larvae. However, the higher doses used to assess threshold toxicity levels caused significant mortality. Our data suggest that biopesticides are not directly harmful to amphibian embryos or larvae in concentrations regularly applied for pest control. Environ Toxicol Chem 2019;39:155-161. © 2019 SETAC.

Assessment of Sublethal Effects of Neonicotinoid Insecticides on the Life-History Traits of 2 Frog Species.

Neonicotinoid insecticides are used extensively in agriculture and, as a consequence, are now detectable in nearby aquatic environments. Few studies have evaluated the effects of neonicotinoids on amphibians in these aquatic environments. In the present study, we examined the effects of 2 commercial formulations of neonicotinoids (active ingredients clothianidin and thiamethoxam) on survival and life-history traits of wood frogs (Lithobates sylvaticus) and northern leopard frogs (Lithobates pipiens). We used artificial pond mesocosms to assess the effects of these neonicotinoids, at nominal concentrations of 2.5 and 250 µg/L, on amphibian larval development through metamorphosis. We found no differences between controls and neonicotinoid exposure for any of the endpoints assessed for either wood frogs or leopard frogs. The present study suggests that concentrations meeting or exceeding observed levels of clothianidin and thiamethoxam in surface waters will not directly affect metamorphosis in 2 amphibians. Environ Toxicol Chem 2019;38:1967-1977. © 2019 SETAC.

Effect of Individual and Combined Treatments of Pesticide, Fertilizer, and Salt on Growth and Corticosterone Levels of Larval Southern Leopard Frogs (Lithobates sphenocephala).

Human activities have introduced a variety of chemicals, including pesticides, fertilizers, and salt, into the environment, which may have deleterious effects on the organisms inhabiting these areas. Amphibians are especially susceptible to absorption of chemical pollutants. To determine the possible combined effects of these chemicals on amphibian development and stress levels, Southern leopard frog (Lithobates sphenocephala) larvae were exposed to one of eight individual or combined treatments of atrazine, ammonium nitrate fertilizer, and sodium chloride salt. Stress levels, indicated by release of the stress hormone corticosterone, were measured premetamorphosis at week 8 of development. Water hormone samples were processed to analyze corticosterone levels. Changes in tadpole growth were determined by surface area measurements taken from biweekly photographs. The combined chemical treatment of atrazine, salt, and fertilizer had a significant interactive effect by increasing stress levels before metamorphosis (p = 0.003). After a month of larval development, tadpoles exposed to ammonium nitrate had larger surface area (p = 0.035). Tadpoles exposed to atrazine had a lower growth rate throughout larval development (p = 0.025) and the lowest number of individuals reaching metamorphosis at 33%. However, the frogs in the atrazine treatment that did successfully metamorphose did so in fewer days (p = 0.002). Because amphibians are exposed to multiple chemicals simultaneously in the environment, assessing the effects of a combination of contaminants is necessary to improve application strategies and ecosystem health.

Insecticide-induced changes in amphibian brains: How sublethal concentrations of chlorpyrifos directly affect neurodevelopment.

Widespread use of pesticides often contaminates natural habitats, exposing nontarget organisms to pesticides that were designed to control pest populations. Even low levels of pesticides can affect aquatic communities both directly and indirectly. Previous work has shown that trace amounts of the pesticide chlorpyrifos altered tadpole morphology and neurodevelopment in artificial ponds (mesocosms). To determine whether effects resulted from direct chlorpyrifos exposure or from disruption of the food web due to a pesticide-induced decline in zooplankton, we examined the impacts of chlorpyrifos on amphibian development in the presence of chlorpyrifos-resistant zooplankton, a key component of the aquatic trophic community. Northern leopard frog (Lithobates pipiens) tadpoles were reared through metamorphosis in mesocosms containing either 0 or 1 µg/L chlorpyrifos and either chlorpyrifos-resistant or chlorpyrifos-sensitive Daphnia pulex zooplankton. Developmental exposure to chlorpyrifos resulted in metamorphs with a relatively wider optic tectum, medulla, and diencephalon compared with controls, and this result was found regardless of the zooplankton population within the mesocosm. Thus, chlorpyrifos directly impacted brain development, independent of the effects on the trophic community. With respect to body shape, chlorpyrifos had no effect on body shape of metamorphs reared in mesocosms with chlorpyrifos-sensitive zooplankton, but body shape was sensitive to zooplankton population in the absence of chlorpyrifos. To conclude, low, ecologically relevant doses of organophosphorous pesticides can directly impact neurodevelopment in a vertebrate model. Environ Toxicol Chem 2018;37:2692-2698. © 2018 SETAC.

Effects of chemical management for invasive plants on the performance of Lithobates pipiens tadpoles.

Invasive plants impact amphibians by altering habitat, altering species interactions, and releasing potentially toxic secondary chemicals. Despite being costly and having the potential to affect nontarget wildlife, chemical management is commonly used to control invasive plants. Prior research has indicated that individual effects of invasive plants or herbicides can be harmful to aquatic organisms; however, information is lacking on the combined effect of these factors on amphibians. A laboratory experiment was performed to assess the impact of leachates of the invasive plants Eurasian watermilfoil (Myriophyllum spicatum) and European buckthorn (Rhamnus cathartica), the herbicide Renovate® 3 (triclopyr [3, 5, 6-trichloro-2-pyridinyloxyacetic acid]), and the combined effects of each plant leachate and the herbicide on the growth, morphology, and survival of northern leopard frog (Lithobates pipiens) tadpoles. No effects of treatment on survival were observed. Tadpole exposure to M. spicatum reduced body mass by 17%, exposure to R. cathartica increased body mass by 36%, and exposure to R. cathartica + low herbicide increased body mass by 38% (although only early in the experiment). Exposure to Renovate 3 induced a 16% and 29% decrease in tadpole size in lower (0.22 mg triclopyr active ingredient [a.i.]/L) and higher (0.92 mg triclopyr a.i./L) concentration treatments, respectively. Results from the present study highlight the importance of considering both individual and combined effects of invasive plants and herbicides because they may have different outcomes for tadpole growth and development. Environ Toxicol Chem 2017;36:2958-2964. © 2017 SETAC.

Dietary antioxidants enhance immunocompetence in larval amphibians.

Dietary antioxidants have been shown to confer a variety of benefits through their ability to counter oxidative stress, including increased immunocompetence and reduced susceptibility to both infectious and non-infectious diseases. However, little is known about the effects of dietary antioxidants on immune function in larval amphibians, a group experiencing worldwide declines driven by factors that likely involve altered immunocompetence. We investigated the effects of dietary antioxidants (quercetin, vitamin E, and ß-carotene) on two components of the immune system, as well as development and growth. Lithobates pipiens tadpoles fed diets with supplemental ß-carotene or vitamin E exhibited an enhanced swelling response as measured with a phytohemagglutinin assay (PHA), but there was no induced antibody response. Effects were often dose-dependent, with higher antioxidant levels generally conferring stronger swelling that possibly corresponds to the innate immune response. Our results indicate that the antioxidant content of the larval amphibian diets not only had a detectable effect on their immune response capability, but also promoted tadpole growth (mass gain), although developmental stage was not affected. Given that many environmental perturbations may cause oxidative stress or reduce immunocompetence, it is critical to understand how nutrition may counter these effects.

Copper and nickel effects on survival and growth of northern leopard frog (Lithobates pipiens) tadpoles in field-collected smelting effluent water.

Trace metals can have subtle yet chronic impacts on organisms by inducing physiological stress that reduces their survival or impedes their ability to tolerate additional environmental stressors. The toxicity literature indicates, however, that aquatic organisms react differently to trace metals depending on the environments in which they reside. The objective of the present study was to understand the response of northern leopard frog (Lithobates pipiens) larvae to ionic copper (Cu), nickel (Ni), and their combination within an effluent water collected downstream of a tailings wetland area. Tadpoles were assigned randomly to 1 of 8 Cu concentrations (8-200 µg/L), 7 Ni concentrations (160-1200 µg/L), or 8 Cu and Ni combined concentrations (8:160-200:1200 µg/L) and showed significant differences in survival and life history traits among treatments. In the Cu and Cu and Ni combined treatments, tadpole survival decreased with increased Cu exposure starting at Cu = 160 µg/L and in the Ni treatment, tadpole survival decreased with increased Ni exposure starting at Ni = 650 µg/L. All Cu-exposed treatments induced a growth increase as the concentration increased, whereas the tadpoles showed a significant decrease in growth rate in Ni treatments. These contrasting outcomes suggest a plastic response to trace metals whereby tadpoles allocate energy reserves toward either escaping or coping with stress. Finally, the authors' argue that future studies will benefit from examining the impacts of multiple stressors in aquatic ecosystems to provide better environmental mitigation.

Helminth community structure in tadpoles of northern leopard frogs (Rana pipiens) and Woodhouse's toads (Bufo woodhousii) from Nebraska.

Currently, little information is available on helminth community structure in tadpoles, the ephemeral, feeding, non-reproductive larva stages of anurans. We examined the helminth community structure in tadpoles of northern leopard frogs, Rana pipiens, and Woodhouse's toads, Bufo woodhousii, from a first-order stream in Western Nebraska. Specifically, we were interested in how species-specific difference in size, time to metamorphosis, and habitat partitioning among larval anurans affect their helminth community structure. From June-July 2009, we collected 30 tadpoles of northern leopard frogs, R. pipiens, and 50 tadpoles of Woodhouse's toads, B. woodhousii, from Cedar Creek, Keith County, NE, USA. The compound helminth community was dominated by trematode metacercarial stages (echinostomatids and plagiorchids) and the nematode Gyrinicola batrachiensis with only a single non-gravid adult trematode, Gorgoderina attenuata, present in low numbers. The helminth component communities were depauperate and isolationist in nature, with a maximum of four helminth species/types per component community. Although no species-specific helminth infections were observed, clear differences were found in overall abundance of nematode versus trematode infections among tadpoles of these two anuran species. The component community of tadpoles of R. pipiens was dominated by nematodes, whereas the component community of tadpoles of B. woodhousii was dominated by trematodes. Although differences in tadpole susceptibility of these two anuran species could not be ruled out, differences in the reproductive strategy of G. batrachiensis in tadpoles of R. pipiens and B. woodhousii and anuran species-specific habitats partitioning among tadpoles of these two anuran species were important factors in structuring their helminth communities. Our study indicates that time to metamorphosis (older tadpoles) play a significant role in structuring helminth communities of tadpoles within species at the infracommunity level with older tadpoles having higher helminth species richness and/or abundances. However, species-specific differences in anuran tadpole habitat partitioning outweighed the effects of differences in time to metamorphosis at the component community level. Finally, our study is the first to report deformities in hind limb development in a tadpole from Nebraska. Of the 30 northern leopard frog tadpoles collected, a single individual contained an extra hind limb and was infected with two plagiorchid metacercariae located in the musculature above the hind limbs.

The effects of a fungicide and chytrid fungus on anuran larvae in aquatic mesocosms.

The amphibian disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), has been linked to significant amphibian declines over the past three decades. The most severe effects of the pathogen have been primarily observed in relatively pristine areas that are not affected by many anthropogenic factors.One hypothesis concerning improved amphibian persistence with Bd in disturbed landscapes is that contaminants may abate the effects of Bd on amphibians. Recent laboratory studies have shown that pesticides, specifically the fungicide thiophanate-methyl (TM), can kill Bd outside of hosts and clear Bd infections within hosts. Using aquatic mesocosms, we tested the hypothesis that TM (0.43 mg/L) would alter growth and development of Lithobates sphenocephalus (southern leopard frog) tadpoles and Bd-infection loads in infected individuals. We hypothesized that the scope of such alterations and infection clearing would be affected by aquatic community variables, specifically zooplankton. TM altered zooplankton diversity (reduced cladoceran and increased copepod and ostracod abundances) and caused mortality to all tadpoles in TM-exposed tanks. In TM-free tanks, Bd-exposed tadpoles in high-density treatments metamorphosed smaller than Bd-unexposed, effects that were reversed in low-density treatments. Our study demonstrates the potential adverse effects of a fungicide and Bd on tadpoles and aquatic systems.

Cranial muscle development in frogs with different developmental modes: direct development versus biphasic development.

Normal development in anurans includes a free swimming larva that goes through metamorphosis to develop into the adult frog. We have investigated cranial muscle development and adult cranial muscle morphology in three different anuran species. Xenopus laevis is obligate aquatic throughout lifetime, Rana(Lithobates) pipiens has an aquatic larvae and a terrestrial adult form, and Eleutherodactylus coqui has direct developing juveniles that hatch from eggs deposited on leaves (terrestrial). The adult morphology shows hardly any differences between the investigated species. Cranial muscle development of E. coqui shows many similarities and only few differences to the development of Rana (Lithobates) and Xenopus. The differences are missing muscles of the branchial arches (which disappear during metamorphosis of biphasic anurans) and a few heterochronic changes. The development of the mandibular arch (adductor mandibulae) and hyoid arch (depressor mandibulae) muscles is similar to that observed in Xenopus and Rana (Lithobates), although the first appearance of these muscles displays a midmetamorphic pattern in E. coqui. We show that the mix of characters observed in E. coqui indicates that the larval stage is not completely lost even without a free swimming larval stage. Cryptic metamorphosis is the process in which morphological changes in the larva/embryo take place that are not as obvious as in normal metamorphosing anurans with a clear biphasic lifestyle. During cryptic metamorphosis, a normal adult frog develops, indicating that the majority of developmental mechanisms towards the functional adult cranial muscles are preserved.

Immunomodulation in post-metamorphic northern leopard frogs, Lithobates pipiens, following larval exposure to polybrominated diphenyl ether.

Pollutants and disease are factors implicated in amphibian population declines, and it is hypothesized that these factors exert a synergistic adverse effect, which is mediated by pollutant-induced immunosuppression. Polybrominated diphenyl ethers (PBDEs) are ubiquitous pollutants that can exert immunotoxicity, making them of interest to test effects on amphibian immune function. We orally exposed Lithobates (Rana) pipiens tadpoles to environmentally realistic levels (0-634 ng/g wet diet) of a pentabromodiphenyl ether mixture (DE-71) from as soon as they became free-swimming through metamorphic climax. To assess adaptive immune response in juvenile frogs, we used an enzyme-linked immunosorbent assay to measure specific IgY production following immunization with keyhole limpet hemocyanin (KLH). Specific KLH antibody response was significantly decreased in juvenile frogs that had been exposed to PBDEs as tadpoles. When assessing innate immune responses, we found significantly different neutrophil counts among treatments; however, phagocytic activity of neutrophils was not significantly different. Secretion of antimicrobial skin peptides (AMPs) nonsignificantly decreased with increasing PBDE concentrations, and no significant effect of PBDE treatment was observed on efficacy of AMPs to inhibit chytrid fungus (Batrachochytrium dendrobatidis) growth. Our findings demonstrate that environmentally realistic concentrations of PBDEs are able to alter immune function in frogs; however, further research is needed to determine how these alterations impact disease susceptibility in L. pipiens.

Apoptosis in larval and frog skin of Rana pipiens, R. catesbeiana, and Ceratophrys ornata.

Apoptosis (programmed cell death) occurs during normal development of anurans in organs such as gills, gut, and tail. For example, apoptotic cells have been reported in the luminal epithelium along the length of the digestive tract of both larvae and frogs; however, timing of the peak number of such cells varies in different species. The purpose of the present study was to ascertain whether apoptosis also varies by species during metamorphic restructuring of the skin (as larval epithelium is replaced by adult epidermis). To determine this, cross-sections of dorsal skin from representative larval stages and frogs of Rana pipiens, R. catesbeiana, and Ceratophrys ornata were incubated with monoclonal antibody against active caspase-3, one of the main enzymes in the apoptotic cascade. We observed apoptotic cells in the epidermis of the skin of the three species and found that such cells were more numerous in larval stages than in frogs and more abundant in the two ranid species than in C. ornata. These results contribute to our understanding of metamorphic changes in anuran skin.

Interactive effects of competition and predator cues on immune responses of leopard frogs at metamorphosis.

Recent hypotheses suggest that immunosuppression, resulting from altered environmental conditions, may contribute to the increased incidence of amphibian disease around the world. Antimicrobial peptides (AMPs) in amphibian skin are an important innate immune defense against fungal, viral and bacterial pathogens. Their release is tightly coupled with release of the stress hormone noradrenaline (norepinephrine). During metamorphosis, AMPs may constitute the primary immune response in the skin of some species because acquired immune functions are temporarily suppressed in order to prevent autoimmunity against new adult antigens. Suppression of AMPs during this transitional stage may impact disease rates. We exposed leopard frog tadpoles (Lithobates pipiens) to a factorial combination of competitor and caged-predator environments and measured their development, growth and production of hydrophobic skin peptides after metamorphosis. In the absence of predator cues, or if the exposure to predator cues was late in ontogeny, competition caused more than a 250% increase in mass-standardized hydrophobic skin peptides. Predator cues caused a decrease in mass-standardized hydrophobic skin peptides when the exposure was late in ontogeny under low competition, but otherwise had no effect. Liquid chromatography tandem mass spectrometry of the skin peptides showed that they include six AMPs in the brevinin and temporin families and at least three of these peptides are previously uncharacterized. Both of these peptide families have previously been shown to inhibit harmful microbes including Batrachochytrium dendrobatidis, the fungal pathogen associated with global amphibian declines. Our study shows that amphibians may be able to adjust their skin peptide defenses in response to stressors that are experienced early in ontogeny and that these effects extend through an important life-history transition.

Investment into defensive traits by anuran prey (Lithobates pipiens) is mediated by the starvation-predation risk trade-off.

Prey can invest in a variety of defensive traits when balancing risk of predation against that of starvation. What remains unknown is the relative costs of different defensive traits and how prey reconcile investment into these traits when energetically limited. We tested the simple allocation model of prey defense, which predicts an additive effect of increasing predation risk and resource availability, resulting in the full deployment of defensive traits under conditions of high risk and resource saturation. We collected morphometric, developmental, and behavioural data in an experiment using dragonfly larvae (predator) and Northern leopard frog tadpoles (prey) subject to variable levels of food availability and predation risk. Larvae exposed to food restriction showed limited response to predation risk; larvae at food saturation altered behaviour, development, and growth in response to predation risk. Responses to risk varied through time, suggesting ontogeny may affect the deployment of particular defensive traits. The observed negative correlation between body size and activity level for food-restricted prey--and the absence of a similar response among adequately-fed prey--suggests that a trade-off exists between behavioural and growth responses when energy budgets are limited. Our research is the first to demonstrate how investment into these defensive traits is mediated along gradients of both predation risk and resource availability over time. The interactions we demonstrate between resource availability and risk level on deployment of inducible defenses provide evidence that both internal condition and extrinsic risk factors play a critical role in the production of inducible defenses over time.

Effects of host species and life stage on the helminth communities of sympatric northern leopard frogs (Lithobates pipiens) and wood frogs (Lithobates sylvaticus) in the Sheyenne National Grasslands, North Dakota.

We studied helminth communities in sympatric populations of leopard frogs (Lithobates pipiens) and wood frogs (Lithobates sylvaticus) and assessed the effects of host species and life stage on helminth community composition and helminth species richness. We examined 328 amphibians including 218 northern leopard frogs and 110 wood frogs collected between April and August of 2009 and 2010 in the Sheyenne National Grasslands of southeastern North Dakota. Echinostomatid metacercariae were the most common helminths found, with the highest prevalence in metamorphic wood frogs. Host species significantly influenced helminth community composition, and host life stage significantly influenced the component community composition of leopard frogs. In these sympatric populations, leopard frogs were common hosts for adult trematodes whereas wood frogs exhibited a higher prevalence of nematodes with direct life cycles. Metamorphic frogs were commonly infected with echinostomatid metacercariae and other larval trematodes whereas juvenile and adult frogs were most-frequently infected with directly transmitted nematodes and trophically transmitted trematodes. Accordingly, helminth species richness increased with the developmental life stage of the host.

Unlikely remedy: fungicide clears infection from pathogenic fungus in larval southern leopard frogs (Lithobates sphenocephalus).

Amphibians are often exposed to a wide variety of perturbations. Two of these, pesticides and pathogens, are linked to declines in both amphibian health and population viability. Many studies have examined the separate effects of such perturbations; however, few have examined the effects of simultaneous exposure of both to amphibians. In this study, we exposed larval southern leopard frog tadpoles (Lithobates sphenocephalus) to the chytrid fungus Batrachochytrium dendrobatidis and the fungicide thiophanate-methyl (TM) at 0.6 mg/L under laboratory conditions. The experiment was continued until all larvae completed metamorphosis or died. Overall, TM facilitated increases in tadpole mass and length. Additionally, individuals exposed to both TM and Bd were heavier and larger, compared to all other treatments. TM also cleared Bd in infected larvae. We conclude that TM affects larval anurans to facilitate growth and development while clearing Bd infection. Our findings highlight the need for more research into multiple perturbations, specifically pesticides and disease, to further promote amphibian heath.

Effects of chronic copper exposure on development and survival in the southern leopard frog (Lithobates [Rana] sphenocephalus).

Exposure to environmental contaminants contributes to the global decline of amphibian populations. The impacts of organic contaminants on amphibians are well documented. However, substantially less is known concerning the potential effects of metals on amphibian populations. Copper (Cu) is an essential element, but it can be toxic at concentrations only slightly higher than the normal physiological range. The present study examines the effects of chronic Cu exposure on embryos and larvae of southern leopard frogs, Lithobates (Rana) sphenocephalus. Groups of eggs from multiple clutches were collected from two wetlands and exposed to a range of Cu concentrations (0-150 µg/L) until they reached the free-swimming stage, and then individual larvae were reared to metamorphosis. Higher Cu concentrations significantly reduced embryo survival to the free-swimming stage but did not further reduce survival to metamorphosis. Larval period was affected by Cu treatment, but the clutch from which larvae originated (i.e., parentage) explained a higher proportion of the variation. Embryo survival to hatching varied significantly among clutches, ranging from 42.9 to 79.2%. Measurable levels of Cu were found in larvae with body burdens up to 595 µg Cu/g dry mass in the 100 µg/L treatment, and larval Cu body burdens were higher than in metamorphs. The present study also demonstrated that higher initial egg density ameliorated embryo mortality at higher Cu levels and should be accounted for in future studies.

Developmental profiles and thyroid hormone regulation of brain transcripts in frogs: a species comparison with emphasis on Physalaemus pustulosus.

In amphibians, thyroid hormones (THs) are considered key regulators of brain remodeling during metamorphosis, while sex steroids (estrogens and androgens) control sexual differentiation and gonadal development. However, these two endocrine axes can interact during tadpole brain development. Previously, we demonstrated that THs affect sex steroid-related gene expression in the developing brain of Silurana tropicalis and Rana pipiens; however, the gene expression changes differed between species. We chose to study a third anuran species, Physalaemus pustulosus, to test new hypotheses about the role of THs in the regulation of brain gene expression. We first established developmental transcript profiles of TH- and sex steroid-related genes in the brain of P. pustulosus. Then, following the same protocols as in our previous studies, we investigated triiodothyronine (T3) regulation of brain transcripts in premetamorphic P. pustulosus and then compared the results with our previous two studies. In the case of TH-related genes, TH receptor beta (trbeta) and deiodinase type 3 (dio3), mRNA developmental profiles were similar in the three species and with respect to other species in the published literature. However, the profiles of TH receptor alpha (tralpha) and deiodinase type 2 (dio2) mRNA revealed differences between anuran species. Among the three anurans we have studied, the direction of the T3 regulation of TH-related genes was overall similar, but the magnitude of gene expression change differed depending on the rate of metamorphosis in a given species. For the sex steroid-related genes, each species exhibited similar developmental profiles but differed in their response to T3. In P. pustulosus, T3 reduced the expression of aromatase (cyp19) while increasing mRNA levels of androgen and estrogen receptors. These results are similar to previous research in R. pipiens but differ from data for S. tropicalis, for which we found an increase in androgen synthesis enzymes but no effect on cyp19. Together, we propose that T3 has the potential to induce the brain androgen system in anurans. This could be achieved by increasing androgen synthesis enzymes (S. tropicalis) or by decreasing estrogen synthesis (due to a decrease in cyp19 in P. pustulosus and R. pipiens). In conclusion, we demonstrated that mechanisms of hormone interactions differ between anuran species, but in all cases T3 appears to affect the balance of sex steroids in the brain, stimulating the androgen system. We have shown that the regulation of sex steroid-related genes by T3 is more similar among closely related species than species with similar reproductive and developmental characteristics.