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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

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., (© 2022 The Ecological Society of America.)

Amphibians are declining globally. Exposure to pesticides has been implicated in decreasing amphibian immune function, thus increasing their susceptibility to parasites and disease and thereby negatively affecting individuals and populations. Amphibians are likely exposed to neonicotinoids because these widely used insecticides are highly soluble in water and because amphibian freshwater habitats are often embedded in agroecosystems. Herein, we investigate the effects of long-term exposure to two individual neonicotinoids (clothianidin or thiamethoxam) at either low or high concentrations (2.5 or 250 µg/L) on northern leopard frog (Lithobates pipiens) blood cell profiles and concentrations of corticosterone, an energy-mediating hormone associated with stress. Larval frogs from Gosner stage 25 to 46 were exposed to pesticide and control treatments in outdoor mesocosms. Corticosterone concentrations were measured after 6 d of exposure, and blood cell profiles were assessed once frogs reached Gosner stage 46 (following 8 w of exposure). No significant changes were found in erythrocyte counts, leukocyte counts, monocyte to leukocyte ratios or corticosterone concentrations between treatments. However, exposure to either 2.5 or 250 µg/L of clothianidin, or 250 µg/L of thiamethoxam decreased neutrophil to lymphocyte ratios and neutrophil to leukocyte ratios, and exposure to 2.5 µg/L of clothianidin or 250 µg/L of thiamethoxam decreased eosinophil to leukocyte ratios. Our results indicate that long-term exposure to neonicotinoids can alter leukocyte profiles, indicative of a stress response. Future studies should investigate whether chronic exposure to neonicotinoids affect multiple measures of stress differently or influences the susceptibility of amphibians to parasites and pathogens. Our work underscores the importance of continued use of multiple measures of stress for different amphibian species when undertaking ecotoxicological assessments., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

As a free-living larval stage of a vertebrate, tadpoles are good subjects for the study of the development of physiological systems and the study of evolutionarily conserved, context-dependent responses to variable environments. While the basic components of innate and adaptive immune defenses in tadpoles are known, the impact of glucocorticoids on immune defenses in tadpoles is not well-studied. We completed four experiments to assess effects of elevation of corticosterone on humoral innate defenses and antibody-mediated immunity in southern leopard frog tadpoles (Lithobates sphenocephalus). To test humoral innate defense within the tadpoles exposed to short-term and long-term elevation of glucocorticoids, we exposed tadpoles to exogenous corticosterone for different lengths of time in each experiment (0-84 days). We used bacterial killing assays to assess humoral innate immune defense. To test antibody-mediated immune responses, we again exposed tadpoles to exogenous corticosterone, while also exposing them to Aeromonas hydrophila. We used A. hydrophila ELISA comparing IgM and IgY responses among groups. Plasma from corticosterone-dosed tadpoles killed more A. hydrophila than control tadpoles each following a short-term (14 day) and long-term (56 day) exposure to exogenous corticosterone. Conversely, corticosterone-dosed tadpoles had significantly lower IgM and IgY against A. hydrophila after 12 weeks. Our fourth experiment revealed that the lower IgY response is a product of weaker, delayed isotype switching compared with controls. These results show that elevated corticosterone has differential effects on innate and acquired immunity in larval southern leopard frogs, consistent with patterns in more derived vertebrates and in adult frogs., (© 2020 Wiley Periodicals LLC.)

Anuran amphibians are common model organisms in bioacoustics and neurobiology. To date, however, most available methods for studying auditory processing in frogs are highly invasive and thus do not allow for longitudinal study designs, nor do they provide a global view of the brain, which substantially limits the questions that can be addressed. The goal of this study was to identify areas in the frog brain that are responsible for auditory processing using in vivo manganese-enhanced MRI (MEMRI). We were interested in determining if the neural processing of socially relevant acoustic stimuli (e.g., species-specific calls) engages a specific pattern of brain activation that differs from patterns elicited by less- or nonrelevant acoustic signals. We thus designed an experiment, in which we presented three different types of acoustic stimuli (species-specific calls, band-limited noise, or silence) to fully awake northern leopard frogs ( Rana pipiens ) and then conducted MEMRI T1-weighted imaging to investigate differences in signal intensity due to manganese uptake as an indication of brain activity across all three conditions. We found the greatest change in signal intensity within the torus semicircularis (the principal central auditory region), the habenula, and the paraphysis of frogs that had been exposed to conspecific calls compared with noise or silence conditions. Stimulation with noise did not result in the same activation patterns, indicating that signals with contrasting social relevance are differentially processed in these areas of the amphibian brain. MEMRI provides a powerful approach to studying brain activity with high spatial resolution in frogs. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Differences in passive muscle mechanical properties between amphibians and mammals have led to differing hypotheses on the functional role of titin in skeletal muscle. Early studies of frog muscle clearly demonstrated intracellular load bearing by titin, but more recent structural and biological studies in mice have shown that titin may serve other functions. Here, we present biomechanical studies of isolated frog and mouse fibers, and fiber bundles to compare the relative importance of intracellular versus extracellular load bearing in these species. Mouse bundles exhibited increased modulus compared with fibers on the descending limb of the length-tension curve, reaching a 2.4-fold elevation at the longest sarcomere lengths. By contrast, frog fibers and bundles had approximately the same modulus at all sarcomere lengths tested. These findings suggest that in the mouse, both muscle fibers and the ECM are involved in bearing whole muscle passive tension, which is distinct from the load bearing process in frog muscle, where titin bears the majority of whole muscle passive tension., Competing Interests: Competing interestsThe authors declare no competing or financial interests. Author contributionsConceptualization: G.M., R.L.L.; Methodology: G.M., R.L.L.; Software: G.M.; Validation: G.M.; Formal analysis: G.M., R.L.L.; Investigation: G.M.; Data curation: G.M., R.L.L.; Writing - original draft: R.L.L.; Writing - review & editing: G.M., R.L.L.; Visualization: G.M., R.L.L.; Supervision: R.L.L.; Project administration: R.L.L.; Funding acquisition: R.L.L., (© 2018. Published by The Company of Biologists Ltd.)

Cryptic species are a challenge for systematics, but their elucidation also may leave critical information gaps about the distribution, conservation status, and behavior of affected species. We use the leopard frogs of the eastern U.S. as a case study of this issue. We refined the known range of the recently described Rana kauffeldi, the Atlantic Coast Leopard Frog, relative to the region's two other leopard frog species, conducted assessments of conservation status, and improved methods for separating the three species using morphological field characters. We conducted over 2,000 call and visual surveys and took photographs of and tissue samples from hundreds of frogs. Genetic analysis supported a three-species taxonomy and provided determinations for 220 individual photographed frogs. Rana kauffeldi was confirmed in eight U.S. states, from North Carolina to southern Connecticut, hewing closely to the Atlantic Coastal Plain. It can be reliably differentiated in life from R. pipiens, and from R. sphenocephala 90% of the time, based on such characters as the femoral reticulum patterning, dorsal spot size and number, and presence of a snout spot. However, the only diagnostic character separating R. kauffeldi from R. sphenocephala remains the breeding call described in 2014. Based on our field study, museum specimens, and prior survey data, we suggest that R. kauffeldi has declined substantially in the northern part of its range, but is more secure in the core of its range. We also report, for the first time, apparent extirpations of R. pipiens from the southeastern portion of its range, previously overlooked because of confusion with R. kauffeldi. We conclude with a generalized ecological research agenda for cryptic species. For R. kauffeldi, needs include descriptions of earlier life stages, studies of niche partitioning with sympatric congeners and the potential for hybridization, and identification of conservation actions to prevent further declines., Competing Interests: EK and DPQ are affiliated with Hudsonia, Ltd. and CTHerpConsultant, LLC, respectively. The commercial affiliations of these two authors did not play any role in the study. There are no patents or products in development to declare. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

The objective was to better understand how a series compliance alters contraction kinetics and power output of muscle to enhance the work done on a load. A mathematical model was created in which a gravitational point load was connected via a linear spring to a muscle (based on the contractile properties of the sartorius of leopard frogs, Rana pipiens). The model explored the effects of load mass, tendon compliance, and delay between onset of contraction and release of the load (catch) on lift height and power output as measures of performance. Series compliance resulted in increased lift height over a relatively narrow range of compliances, and the effect was quite modest without an imposed catch mechanism unless the load was unrealistically small. Peak power of the muscle-tendon complex could be augmented up to four times that produced with a muscle alone, however, lift height was not predicted by peak power. Rather, lift height was improved as a result of the compliance synchronizing the time courses of muscle force and shortening velocity, in particular by stabilizing shortening velocity such that muscle power was sustained rather than rising and immediately falling. With a catch mechanism, enhanced performance resulted largely from energy storage in the compliance during the period of catch, rather than increased time for muscle activation before movement commenced. However, series compliance introduced a trade-off between work done before versus after release of the catch. Thus, the ability of tendons to enhance locomotor performance (i.e. increase the work done by muscle) appears dependent not only on their established role in storing energy and increasing power, but also on their ability to modulate the kinetics of muscle contraction such that power is sustained over more of the contraction, and maximizing the balance of work done before versus after release of a catch.

Gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone release to control ovulation and spermiation in vertebrates. Dopamine (DA) has a clear inhibitory role in the control of reproduction in numerous teleosts, and emerging evidence suggests that similar mechanisms may exist in amphibians. The interactions between GnRH and DA on spawning success and pituitary gene expression in the Northern leopard frog (Lithobates pipiens) were therefore investigated. Frogs were injected during the natural breeding season with a GnRH agonist [GnRH-A; (Des-Gly 10 , D-Ala 6 , Pro-NHEt 9 )-LHRH; 0.1μg/g and 0.4μg/g] alone and in combination with the dopamine receptor D2 antagonist metoclopramide (MET; 5μg/g and 10μg/g). Injected animals were allowed to breed in outdoor mesocosms. Time to amplexus and oviposition were assessed, and egg mass release, incidences of amplexus, egg mass weight, total egg numbers and fertilization rates were measured. To examine gene expression, female pituitaries were sampled at 12, 24 and 36h following injection of GnRH-A (0.4μg/g) alone and in combination with MET (10μg/g). The mRNA levels of the genes lhb, fshb, gpha, drd2 and gnrhr1 were measured using quantitative real-time PCR. Data were analyzed by a two-way ANOVA. Both GnRH-A doses increased amplexus, oviposition and fertilization alone. Co-injection of MET with GnRH-A did not further enhance spawning success. Injection of GnRH-A alone time-dependently increased expression of lhb, fshb, gpha and gnrhr1. The major effect of MET alone was to decrease expression of drd2. Importantly, the stimulatory effects of GnRH-A on lhb, gpha and gnrhr1 were potentiated by the co-injection of MET at 36h. At this time, expression of fshb was increased only in animals injected with both GnRH-A and MET. Spawning success was primarily driven by the actions of GnRH-A. The hypothesized inhibitory action of DA was supported by pituitary gene expression analysis. The results from this study provide a fundamental framework for future time- and dose-response investigations to improve current spawning methods in amphibians., (Copyright © 2017. Published by Elsevier Inc.)

The Chiricahua leopard frog (Lithobates chiricahuensis) is in decline throughout the western United States, and is particularly sensitive to physical, chemical and biotic changes in their habitat. Acute toxicity tests revealed that among the metals detected in Chiricahua leopard frog habitat, copper was toxic at concentrations lower than those observed in the environment. Developing tadpoles were chronically exposed for 60 days to cadmium, copper and zinc because of the potential for long term exposure to these metals during early development. Cadmium was toxic, but at concentrations above observed environmental levels. Copper was especially toxic to this species at concentrations of about 10% of concentrations observed in their habitats. The onset of toxicity occurred within a few days of exposure, thus pulsed exposures from rain events could potentially be acutely toxic to tadpoles of this species. Zinc did not appear to have a negative impact during the acute or chronic exposures.

New explosive formulations are being developed to be less sensitive to impact and inadvertent explosion, increasing safety for the warfighter. Since testing and training make environmental releases imminent, the toxicity of 3-nitro-1,2,4-triazol-5-one (NTO), a component of Insensitive Munitions eXplosive (IMX) formulations, was assessed in a one-generation study to the northern leopard frog (Lithobates ( = Rana) pipiens). Because NTO in water creates acidic conditions, acute studies were conducted with non-pH-adjusted NTO, while a long-term (70-d) study was conducted with neutralized NTO. In the acute study, 48-h and 7-d LC 50 s were ~250 mg NTO/L. In the long-term study, tadpoles were dead by day 2 in 11,350 mg/L NTO, and by day 63 in 8382 mg/L. The 70-d LC 50 was 3670 mg (neutralized) NTO/L. The number of organisms reaching complete metamorphosis was reduced by NTO; the lowest IC 25 was 1999 mg NTO/L for the Number Completing Metamorphosis. The NOECs for Time to Front Limb Eruption or Time to Metamorphosis were the same at 1346 mg/L. Histopathology did not significantly distinguish between NTO-exposed and unexposed animals, although possible effects on the density of spermatogonia in NTO-exposed males was suggested. The test data indicate that acute toxicity to ambient NTO can be attributed primarily to its acidic nature; relatively low chronic toxicity of neutralized NTO is due to delays in metamorphosis. The consequence from this latter observation may be ecologically significant as delays of even a few days could increase mortality through predation and/or loss of the aquatic medium in temporary water bodies.

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 β-carotene) on two components of the immune system, as well as development and growth. Lithobates pipiens tadpoles fed diets with supplemental β-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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We used physiological and anatomical methods to elucidate how the visual field is represented in the part of the dorsal anterior thalamus of the leopard frog that receives direct retinal projections. We recorded extracellularly while presenting visual stimuli, and characterized a physiologically defined region that encompasses the retinal projections as well as an extended zone beyond them. We probed the area systematically to determine if the zone is organized in a visuotopic map: we found that it is not. We found that units in this region respond only to stimuli in the contralateral half of the visual field, which is similar to what is seen in the dorsal lateral geniculate nucleus in mammals. When we backfilled retinal ganglion cells from application of HRP to the anterior thalamus, we found labeled cells only in those parts of the retina corresponding to the contralateral hemifield, confirming our physiological observations., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Many animals use catapult mechanisms to produce extremely rapid movements for escape or prey capture, resulting in power outputs far beyond the limits of muscle. In these catapults, muscle contraction loads elastic structures, which then recoil to release the stored energy extremely rapidly. Many arthropods employ anatomical 'catch mechanisms' to lock the joint in place during the loading period, which can then be released to allow joint motion via elastic recoil. Jumping vertebrates lack a clear anatomical catch, yet face the same requirement to load the elastic structure prior to movement. There are several potential mechanisms to allow loading of vertebrate elastic structures, including the gravitational load of the body, a variable mechanical advantage, and moments generated by the musculature of proximal joints. To test these hypothesized mechanisms, we collected simultaneous 3D kinematics via X-ray Reconstruction of Moving Morphology (XROMM) and single-foot forces during the jumps of three Rana pipiens. We calculated joint mechanical advantage, moment and power using inverse dynamics at the ankle, knee, hip and ilio-sacral joints. We found that the increasing proximal joint moments early in the jump allowed for high ankle muscle forces and elastic pre-loading, and the subsequent reduction in these moments allowed the ankle to extend using elastic recoil. Mechanical advantage also changed throughout the jump, with the muscle contracting against a poor mechanical advantage early in the jump during loading and a higher mechanical advantage late in the jump during recoil. These 'dynamic catch mechanisms' serve to resist joint motion during elastic loading, then allow it during elastic recoil, functioning as a catch mechanism based on the balance and orientation of forces throughout the limb rather than an anatomical catch., (© 2014. Published by The Company of Biologists Ltd.)

Organisms exhibit plasticity in response to their environment, but there is large variation even within populations in the expression and magnitude of response. Maternal influence alters offspring survival through size advantages in growth and development. However, the relationship between maternal influence and variation in plasticity in response to predation risk is unknown. We hypothesized that variation in the magnitude of plastic responses between families is at least partly due to maternal provisioning and examined the relationship between maternal condition, egg provisioning and magnitude of plastic response to perceived predation risk (by dragonfly larvae: Aeshna spp.) in northern leopard frogs (Lithobates pipiens). Females in better body condition tended to lay more (clutch size) larger (egg diameter) eggs. Tadpoles responded to predation risk by increasing relative tail depth (morphology) and decreasing activity (behaviour). We found a positive relationship between morphological effect size and maternal condition, but no relationship between behavioural effect size and maternal condition. These novel findings suggest that limitations imposed by maternal condition can constrain phenotypic variation, ultimately influencing the capacity of populations to respond to environmental change., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

We describe a new cryptic species of leopard frog from the New York City metropolitan area and surrounding coastal regions. This species is morphologically similar to two largely parapatric eastern congeners, Rana sphenocephala and R. pipiens. We primarily use bioacoustic and molecular data to characterize the new species, but also examine other lines of evidence. This discovery is unexpected in one of the largest and most densely populated urban parts of the world. It also demonstrates that new vertebrate species can still be found periodically even in well-studied locales rarely associated with undocumented biodiversity. The new species typically occurs in expansive open-canopied wetlands interspersed with upland patches, but centuries of loss and impact to these habitats give some cause for conservation concern. Other concerns include regional extirpations, fragmented extant populations, and a restricted overall geographic distribution. We assign a type locality within New York City and report a narrow and largely coastal lowland distribution from central Connecticut to northern New Jersey (based on genetic data) and south to North Carolina (based on call data).

We determined how leopard frogs respond to non-moving aspects of the environment. We have discovered that these frogs are attracted to dark, stationary, opaque objects. This attraction depends on the relative reflectance of the object, i.e., the darker the block, the more attractive it is, and the attraction is found under both bright and dim ambient light levels. Larger blocks are more attractive than smaller blocks, but frogs are still attracted to blocks much smaller than themselves. Previous studies have shown that frogs are also attracted to sources of light. Using a choice experiment, we show that the probability a frog will choose a dark object versus a light source depends on the intensity of the light source relative to the intensity of the ambient light. The frog only moves toward a light source when it is at least 20 times brighter than the brightest object in the environment. These findings help to clarify the frog's "phototactic" nature., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Wiley Periodicals, Inc.)

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.

This study evaluated the impacts of Roundup® on tadpole mouthpart damage as a mechanism for reduced growth and developmental rates in Lithobates sphenocephalus (Southern leopard frog) tadpoles. We did not find evidence that Roundup(®) damages larval mouthparts, nor was there a significant relationship between mouthpart damage and either body condition or developmental rate. However, the highest concentration of Roundup® significantly stunted development compared to all other treatments. Although we observed a significant effect of Roundup® on developmental rate, we conclude that mouthpart damage is likely not a mechanism for this life history response.

Neural adaptation, a reduction in the response to a maintained stimulus, is an important mechanism for detecting stimulus change. Contributing to change detection is the fact that adaptation is often stimulus specific: adaptation to a particular stimulus reduces excitability to a specific subset of stimuli, while the ability to respond to other stimuli is unaffected. Phasic cells (e.g., cells responding to stimulus onset) are good candidates for detecting the most rapid changes in natural auditory scenes, as they exhibit fast and complete adaptation to an initial stimulus presentation. We made recordings of single phasic auditory units in the frog midbrain to determine if adaptation was specific to stimulus frequency and ear of input. In response to an instantaneous frequency step in a tone, 28% of phasic cells exhibited frequency specific adaptation based on a relative frequency change (delta-f=±16%). Frequency specific adaptation was not limited to frequency steps, however, as adaptation was also overcome during continuous frequency modulated stimuli and in response to spectral transients interrupting tones. The results suggest that adaptation is separated for peripheral (e.g., frequency) channels. This was tested directly using dichotic stimuli. In 45% of binaural phasic units, adaptation was ear specific: adaptation to stimulation of one ear did not affect responses to stimulation of the other ear. Thus, adaptation exhibited specificity for stimulus frequency and lateralization at the level of the midbrain. This mechanism could be employed to detect rapid stimulus change within and between sound sources in complex acoustic environments.

The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), has been linked to amphibian declines and extinctions worldwide. The pathogen has been found on amphibians throughout eastern North America, but has not been associated with mass die-offs in this region. In this study, we conducted laboratory experiments on the effects of Bd infection in a putative carrier species, Lithobates pipiens, using two estimators of fitness: jumping performance and testes morphology. Over the 8-week study period, peak acceleration during jumping was not significantly different between infected and uninfected animals. Peak velocity, however, was significantly lower for infected animals after 8 weeks. Two measures of sperm production, germinal epithelium depth, and maximum spermatic cyst diameter, showed no difference between infected and uninfected animals. The width, but not length, of testes of infected animals was significantly greater than in uninfected animals. This study is the first to show effects on whole-organism performance of Bd infection in post-metamorphic amphibians, and may have important long-term, evolutionary implications for amphibian populations co-existing with Bd infection.

Background: Amphibian declines are now recognized globally. It is also well known that many anurans do not reproduce easily in captivity, especially when held over long periods, or if they require hibernation before breeding. A simple method to induce spawning and subsequent development of large numbers of healthy tadpoles is therefore required to meet research and conservation goals., Methods: The method is based on simultaneous injection of both female and male leopard frogs, Lithobates pipiens (formerly called Rana pipiens) with a cocktail of a gonadotropin-releasing hormone agonist (GnRH-A) and a dopamine antagonist. We call this the AMPHIPLEX method, which is derived from the combination of the words amphibian and amplexus. Following injection, the animals are thereby induced, and perform amplexus and natural fertilization under captive conditions., Results: We tested combinations of a GnRH agonist with 2 different dopamine antagonists in L. pipiens in the breeding season. The combination of des-Gly(10), D-Ala(6), Pro-NHEt(9)-GnRH (0.4 micrograms/g body weight; GnRH-A) with metoclopramide hydrochloride (10 micrograms/g body weight; MET) or domperidone (DOM) were equally effective, producing 89% and 88% successful spawning, respectively. This yielded more than 44,000 eggs for the 16/18 females that ovulated in the GnRH-A+MET group, and more than 39,000 eggs for the 15/17 females that ovulated in the GnRH-A+DOM group. We further tested the GnRH-A+MET in frogs collected in the wild in late autumn and hibernated for a short period under laboratory conditions, and report a low spawning success (43%). However, GnRH-A priming 24 hours prior to injections of the GnRH-A+MET cocktail in animals hibernated for 5-6 weeks produced out-of-season spawning (89%) and fertilization (85%) comparable to those we observed for in-season spawning. Assessment of age and weight at metamorphosis indicated that L. pipiens tadpoles resulting from out-of-season spawning grew normally and metamorphosed successfully., Conclusion: We provide evidence for successful captive breeding of the leopard frog, L. pipiens. This simple protocol can be used to obtain large numbers of eggs in a predictable, timed manner.

There is evidence that over the last 30 years, there have been mass declines in diverse geographic locations among amphibian populations due to disease outbreaks. Multiple causes have been suggested to explain this increase in disease incidence. Among these, climate changes, environmental pollution and reduced water quality are gaining attention. Indeed, some chemicals of environmental concerns are known to alter the immune system. It is possible that exposure to these pollutants could alter the immune system of anurans and render them more susceptible to different pathogens. In this study, we sampled Rana pipiens in five different sites near St. Lawrence River (Quebec, Canada) during the months of July and September in 2001. Two of these sites were protected areas, in which low levels of pesticides were detected, while the remaining three sites were located in areas with intensive corn and soybeans cultivations. Our results demonstrated that frogs living in agricultural regions are smaller in size and weight than frogs living in areas with lower levels of pesticides at both sampling times. Moreover, we have observed a significant decrease in the number of splenocytes (cellularity) and the phagocytic activity in frogs sampled in impacted sites. Taken together, these results suggest that frogs living in agricultural regions might be more vulnerable to infections and diseases through their smaller size and alteration of their immune system. Our results also contribute to the overall discussion on factors involved in amphibian declines.

Movements of many animals are powered by a combination of active shortening of muscles and recoil of elastic structures. Movements that rely primarily on elastic recoil, such as the explosive locomotor and feeding movements of many ectotherms, have been shown to exhibit lower thermal dependence than muscle-powered movements because elastically powered movements circumvent the effects of temperature on muscle contractile dynamics. To determine if elastic recoil contributes to the ballistic mouth opening and tongue projection of the frog Rana pipiens, we examined feeding kinematics and dynamics of tongue and jaw movements at three ambient temperatures (10, 15, and 25°C) using 6,000 Hz image sequences. We found that ballistic mouth opening exhibits high instantaneous power (up to 1,783 W kg(-1) of muscle mass) and its dynamics have low thermal dependence (Q(10) values of 1.02-1.37), indicating the contribution of elastic recoil. The dynamics of tongue projection are more thermally dependent (Q(10) of 1.35-2.51) and likely involve muscle activity during the movement. Mouth opening during prey transport, as well as tongue retraction and mouth closing showed little evidence of elastic recoil and their dynamics show relatively high thermal dependence (Q(10) of 1.38-1.89). R. pipiens shows lower thermal robustness and power of tongue projection than ballistic-tongued Bufo terrestris which displays complete thermal independence, suggesting that in Rana tongue projection is not as tightly coupled to jaw depression as in toads. We propose that thermal experiments may be useful in probing for elastic mechanisms in other biomechanical systems., (Copyright © 2012 Wiley Periodicals, Inc.)

Naphthenic acids (NAs) have been cited as one of the main causes of the toxicity related to oil sands process-affected materials and have recently been measured in biological tissues (fish). However, adverse effects have not been a consistent finding in toxicology studies on vertebrates. This study set out to determine two factors: 1) whether exposure to commercial NAs (Refined Merichem) resulted in detectable tissue residues in native amphibians (northern leopard frogs, Lithobates pipiens), and 2) whether such exposure would produce clinical or subclinical toxicity. Frogs were kept in NA solutions (0, 20, or 40 mg/L) under saline conditions comparable to that on reclaimed wetlands in the Athabasca oil sands for 28 days. These exposures resulted in proportional NA concentrations in muscle tissue of the frogs, estimated by gas chromatography-mass spectrometry analyses. Detailed studies determined if the increasing concentrations of NAs, and subsequently increased tissue NA levels, caused a proportional compromise in the health of the experimental animals. Physiological investigations included innate immune function, thyroid hormone levels, and hepatic detoxification enzyme induction, none of which differed in response to increased exposures or tissue concentrations of NAs. Body mass did increase in both the salt- and NA-exposed animals, likely related to osmotic pressure and uptake of water through the skin. Our results demonstrate that commercial NAs are absorbed and deposited in muscle tissue, yet they show few negative physiological or toxicological effects on the frogs., (Copyright © 2012. Published by Elsevier B.V.)

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., (Copyright © 2012 SETAC.)

Anuran jumping is one of the most powerful accelerations in vertebrate locomotion. Several species are hypothesized to use a catapult-like mechanism to store and rapidly release elastic energy, producing power outputs far beyond the capability of muscle. Most evidence for this mechanism comes from measurements of whole-body power output; the decoupling of joint motion and muscle shortening expected in a catapult-like mechanism has not been demonstrated. We used high-speed marker-based biplanar X-ray cinefluoroscopy to quantify plantaris muscle fascicle strain and ankle joint motion in frogs in order to test for two hallmarks of a catapult mechanism: (i) shortening of fascicles prior to joint movement (during tendon stretch), and (ii) rapid joint movement during the jump without rapid muscle-shortening (during tendon recoil). During all jumps, muscle fascicles shortened by an average of 7.8 per cent (54% of total strain) prior to joint movement, stretching the tendon. The subsequent period of initial joint movement and high joint angular acceleration occurred with minimal muscle fascicle length change, consistent with the recoil of the elastic tendon. These data support the plantaris longus tendon as a site of elastic energy storage during frog jumping, and demonstrate that catapult mechanisms may be employed even in sub-maximal jumps.

Effects of exposure to contaminants such as pesticides along with exposure to pathogens have been listed as two major contributors to the global crisis of declining amphibian populations. These two factors have also been linked in explanations of the causes of these population declines. We conducted a combined exposure experiment to test the hypothesis that exposure to two agricultural herbicides would increase the susceptibility of post-metamorphic northern leopard frogs (Lithobates pipiens) to the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). We assessed the independent and interactive effects of these exposures on the health and survival of the frogs. Wild-caught frogs underwent a 21-day exposure to a nominal concentration of either 2.1 μg/L atrazine (Aatrex(®) Liquid 480) or 100 μg a.e./L glyphosate (Roundup(®) Original), followed by Bd, and then were observed until 94 days post-initial exposure to the herbicides. Actual levels of atrazine were between 4.28 ± 0.04 μg/L and 1.70 ± 0.26 μg/L while glyphosate degraded from 100 μg a.e./L to approximately 7 μg a.e./L within 6 days of initial exposure to the herbicides. Compared to controls, the glyphosate formulation reduced the snout-vent length of frogs during the pesticide exposure (at Day 21), and the atrazine formulation reduced gain in mass up to Day 94. No treatment affected survival, splenosomatic or hepatosomatic indices, the densities and sizes of hepatic and splenic melanomacrophage aggregates, the density and size of hepatic granulomas, proportions of circulating leucocytes, the ratio of neutrophils to lymphocytes, or the ratio of leucocytes to erythrocytes. Histological assessment of samples collected at Day 94 revealed no evidence of Bd infection in any Bd-exposed frogs, while real-time PCR detected only one case of light infection in a single atrazine- and Bd-exposed frog. Frogs exposed to Bd shed their skin significantly more frequently than Bd-unexposed frogs, which may have helped them resist or clear infection, and could explain why no interaction between the herbicides and Bd was detected. The results suggest that these frogs were resistant to Bd infection and that pre-exposure to the herbicides did not alter this resistance. The effects seen on the growth following herbicide exposure is a concern, as reduced growth can lower the reproductive success and survival of the amphibians., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Polybrominated diphenyl ethers (PBDEs) are bioaccumulative, persistent organic pollutants used as flame retardants in consumer goods. Concentrations of PBDEs in North American wildlife have been increasing for decades and been shown to have estrogenic effects on sexual development. No studies, however, have examined the effects of PBDEs on the sexual development of North American frogs at ecologically relevant concentrations. This study examined the effects of five dietary concentrations of DE-71 (0, 1.1, 6.1, 71.4, and 634 ng ΣPBDEs/g diet), a technical PBDE mixture, on the gonadal development of the northern leopard frog, Rana pipiens. Tadpoles were exposed chronically from the time they became free-swimming until metamorphosis. Frogs were killed either at metamorphic climax or 10 weeks after completing metamorphosis, processed for histology, and examined for alterations in sexual development. The experimental group exposed to PBDEs at 1.1 ng/g had a significantly larger proportion of females compared with the expected 50:50 sex ratio. At 10 weeks post-metamorphosis, male frogs exposed to 6.1 and 71.4 ng/g had significantly smaller testes, but all other measure of gonadal development tested showed no effects. No intersex or increased incidence of gonadal abnormality were detected. These findings indicate that PBDEs may disrupt sexual differentiation in frogs at low, environmentally relevant concentrations., (Copyright © 2011 SETAC.)

Three species of anuran amphibians (Odorrana tormota, Odorrana livida and Huia cavitympanum) have recently been found to detect ultrasounds. We employed immunohistochemistry and confocal microscopy to examine several morphometrics of the inner ear of these ultrasonically sensitive species. We compared morphological data collected from the ultrasound-detecting species with data from Rana pipiens, a frog with a typical anuran upper cut-off frequency of ∼3 kHz. In addition, we examined the ears of two species of Lao torrent frogs, Odorrana chloronota and Amolops daorum, that live in an acoustic environment approximating those of ultrasonically sensitive frogs. Our results suggest that the three ultrasound-detecting species have converged on small-scale functional modifications of the basilar papilla (BP), the high-frequency hearing organ in the frog inner ear. These modifications include: 1. reduced BP chamber volume, 2. reduced tectorial membrane mass, 3. reduced hair bundle length, and 4. reduced hair cell soma length. While none of these factors on its own could account for the US sensitivity of the inner ears of these species, the combination of these factors appears to extend their hearing bandwidth, and facilitate high-frequency/ultrasound detection. These modifications are also seen in the ears of O. chloronota, suggesting that this species is a candidate for high-frequency hearing sensitivity. These data form the foundation for future functional work probing the physiological bases of ultrasound detection by a non-mammalian ear., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 S. Karger AG, Basel.)

The lifespan of herbivorous Rana pipiens larvae is ∼3 months, while that of carnivorous Ceratophrys ornata larvae is only about 2 weeks. During metamorphic climax, the larval gut shortens dramatically, especially in R. pipiens, and its luminal epithelium is replaced by adult-type epithelium. To determine when programmed cell death occurs during the metamorphic restructuring of the gut, we prepared cross-sections of the stomach, small intestine, and large intestine from representative larval stages and from juvenile frogs of both species. The sections were incubated with monoclonal antibody against active caspase-3, one of the key enzymes in the apoptotic cascade. We observed apoptosis in some luminal epithelial cells in each of the three regions of the larval gastrointestinal tract of both species. However, apoptotic cells appeared earlier in larval stages of R. pipiens than C. ornata and few were seen in juvenile frogs of either species. The results demonstrate the occurrence of apoptosis in the metamorphic remodeling of the gut of both R. pipiens larvae and C. ornata larvae., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Progesterone triggers resumption of the first meiotic division in the Rana pipiens oocyte by binding to the N-terminal external loop of the catalytic subunit of Na/K-ATPase, releasing a cascade of lipid second messengers. This is followed by internalization of specific membrane proteins, plasma membrane depolarization and nuclear membrane breakdown, culminating in arrest at second metaphase., Results: Progesterone initiates an increase in phosphoryl potential during the first meiotic division, resulting in the accumulation of high energy protein phosphate by second metaphase arrest. 31P-NMR, with saturation transfer, demonstrates that the phosphocreatine level rises ~2 fold and that the "pseudo" first order rate constant for the creatine kinase reaction falls to ~20% of the control by the onset of nuclear membrane breakdown. 32PO4 pulse-labeling reveals a net increase in phosphorylation of yolk protein phosvitin during this period. The increased yolk protein phosphorylation coincides with internalization of membrane Na/K-ATPase and membrane depolarizatio, Conclusions: These results indicate that progesterone binding to the catalytic subunit of the Na-pump diverts ATP from cation regulation at the plasma membrane to storage of high energy phosphate in yolk protein. Phosvitin serves as a major energy source during fertilization and early cleavage stages and is also a storage site for cations (e.g. Na+, K+, Ca2+, Fe2+/3+) essential for embryonic development., (© 2011 Morrill et al; licensee BioMed Central Ltd.)

Movement of an image on the retina is necessary for the persistence of vision in vertebrates. Leopard frogs (Rana pipiens) do not show any obvious independent eye movements that could sustain perception of stationary objects when the animal itself is stationary. However, video recordings of normal, awake leopard frogs made through a dissecting microscope reveal that the animal's whole head oscillates with an amplitude of 10-100 μm in step with the breathing cycle. The retinal image shifts produced by these breathing movements could ensure continuous perception of the frog's stationary environment., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Northern leopard frogs (Rana pipiens) have been disappearing throughout their range. The causes for extirpations have been elusive, although habitat alteration appears to be a major factor. Pesticides have been implicated in declines in western amphibians, but no mechanism has been identified. We tested the hypothesis that leopard frog tadpoles would be eliminated from insecticide-exposed ponds when they were less abundant than a competitor (American toads, Bufo americanus) by manipulating toad and leopard frog density (16, 50, or 150 of each species) with or without the insecticide carbaryl. Insecticide exposure did not interact with density on either species, indicating that contaminant effects were similar despite drastic differences in competition. The survival of both species decreased with increasing conspecific density, but not heterospecific density. Leopard frogs produced more metamorphs at intermediate densities, whereas toads produced more metamorphs at high density. Leopard frog mass at and time to metamorphosis responded negatively to density. Carbaryl did not affect leopard frog survival but positively affected mass at and time to metamorphosis. In contrast, toad survival was reduced after exposure, but mass at metamorphosis was greater. Pesticide-induced survival reductions may not affect population size if reductions are slight (as in toads, whose fitness may be compensated by increased mass). Management strategies aimed at ameliorating the effects of carbaryl in systems with these two species may not need to consider density., (Copyright © 2010 SETAC.)

Frogs exhibit flexible repertoires of prey-capture behavior, which depend primarily on visual analysis of prey attributes. We review three examples of how visual cues are used to modulate prey-capture strategies. (1) Dyscophus guineti modulates tongue aiming in response to prey location. These frogs turn only their heads to apprehend prey located at azimuths <40°. At azimuths >40°, the frogs switch from this strategy to one in which both head and tongue are aimed toward prey. (2) Rana pipiens modulates its feeding behavior in response to prey size, using tongue prehension for capturing small prey but switching to jaw prehension to capture large prey. (3) In Cyclorana novaehollandiae, visual processing of prey attributes involves hierarchical decision making. These frogs first assess prey size. For large prey, they ignore velocity but not shape. For small prey, they ignore shape but not velocity. Alternative prey-capture strategies are associated with biomechanical trade-offs that result from the interaction between the feeding apparatus and varying attributes of prey. Alternative strategies likely exist because biomechanical constraints prevent any one strategy from being effective over a range of prey attributes. Taken together, these studies emphasize the requirement that predators must somehow tune prey-capture kinematics simultaneously to multiple attributes of prey. In frogs, the choice among alternative prey-capture strategies involves a hierarchical decision-making process. Hierarchical decision making is expected to be widespread among animals. However, no previous studies were found except for humans, who frequently use this type of approach to make complex decisions., (Copyright © 2010 Wiley-Liss, Inc., A Wiley Company.)

An important contributing factor for the high sensitivity of sensory systems is the exquisite sensitivity of the sensory receptor cells. We report here the signaling threshold of the olfactory receptor neuron (ORN). We first obtained a best estimate of the size of the physiological electrical response successfully triggered by a single odorant-binding event on a frog ORN, which was ∼0.034 pA and had an associated transduction domain spanning only a tiny fraction of the length of an ORN cilium. We also estimated the receptor-current threshold for an ORN to fire action potentials in response to an odorant pulse, which was ∼1.2 pA. Thus, it takes about 35 odorant-binding events successfully triggering transduction during a brief odorant pulse in order for an ORN to signal to the brain.

The basilar papilla (BP) in the frog inner ear is a relatively simple auditory receptor. Its hair cells are embedded in a stiff support structure, with the stereovilli connecting to a flexible tectorial membrane (TM). Acoustic energy passing the papilla presumably causes displacement of the TM, which in turn deflects the stereovilli and stimulates the hair cells. In this paper we present optical measurements of the mechanical response of the TM to various stimulus levels. Results were obtained from 3 specimens (4 ears). The phase of the displaced area of the TM was constant across stimulus levels. Phase differences between the orthogonal spatial motion components were either close to 0 degrees or 180 degrees. These findings were consistent with a TM motion along the epithelium surface. The TM response was linear for stimulus levels up to -30 dB (re. 1 microm) at the operculum. This amplitude was estimated to exceed that at which neural responses saturate. Apparently, saturation of the neural response in the frog inner ear is not based on saturation of the mechanical response of the tectorial membrane., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Background: There are conflicting reports regarding the effects of atrazine (ATZ) on amphibian development. Therefore, further studies are needed to examine the potential mechanisms of action of ATZ in amphibians., Objectives: Our aim in this study was to determine whether low concentrations of ATZ affect gonadal development and metamorphosis in the Northern leopard frog, Rana pipiens., Methods: Tadpoles were exposed in outdoor mesocosms to nominal concentrations of 0.1 and 1.8 microg/L of formulated ATZ from Gosner stage 27 (G27) to metamorphic climax (G42). Exposure to 17alpha-ethinylestradiol (EE2; 1.5 microg/L) provided a positive control for induction of testicular oocytes in males. Endocrine-related gene expression and gonadal histopathology were examined at G42 and in a subset of premetamorphic G34 tadpoles that failed to metamorphose., Results: Gonadal gross morphology revealed that the 1.8-microg/L ATZ treatment produced 20% more females compared with the control. Histologic analysis revealed that 22% of EE2-treated males had testicular oocytes, whereas none were observed in any animals from the control or either ATZ groups. ATZ increased brain estrogen receptor alpha mRNA to 2.5 times that of the control at premetamorphosis and altered liver levels of 5beta-reductase activity at metamorphosis. In contrast, brain aromatase mRNA level and activity did not change. ATZ treatments significantly reduced metamorphic success (number of animals reaching metamorphosis) without affecting body weight, snout-vent length, or age at metamorphosis. Gene expression analysis indicated that ATZ decreased the expression of deiodinase type 3 in the tail at premetamorphosis., Conclusions: Our study indicates that exposure to low concentrations of ATZ in experimental mesocosms alters gonadal differentiation and metamorphosis in developing R. pipiens.

The frog nucleus isthmi (parabigeminal nucleus in mammals) is a visually responsive, cholinergic and anatomically well-defined group of neurons in the midbrain. It shares reciprocal topographic projections with the ipsilateral optic tectum (superior colliculus in mammals) and strongly influences visual processing. Anatomical and biochemical information indicates the existence of distinct neural populations within the frog nucleus isthmi, which raises the question: are there electrophysiological distinctions between neurons that are putatively classified by their anatomical and biochemical properties? To address this question, we measured frog nucleus isthmi neuron cellular properties in vitro and visual response properties in vivo. No evidence for distinct electrophysiological classes of neurons was found. We thus conclude that, despite the anatomical and biochemical differences, the cells of the frog nucleus isthmi respond homogeneously to both current injections and simple visual stimuli.

Measurements of laser-stimulated action potentials in the sciatic nerve of leopard frogs (Rana pipiens) are made using two infrared lasers. The dorsal sides of the frog's hind limbs are exposed to short-pulsed 1540- and 1064-nm wavelengths at three separate spot sizes: 2, 3, and 4 mm. Energy density thresholds are determined for eliciting an action potential at each experimental condition. Results from these exposures show similar evoked potential thresholds for both wavelengths. The 2-mm-diam spot sizes yield action potentials at radiant exposure levels almost double that seen with larger beam sizes.

Many organisms endure extended periods of dormancy by depressing their metabolism, which effectively prolongs the use of their endogenous energy stores. Though the mechanisms of hypometabolism are varied and incompletely understood, recent work suggests that urea accumulation in autumn and early winter contributes to reduced metabolism of hibernating wood frogs (Rana sylvatica). Urea accumulation during dormancy is a widespread phenomenon, and it has long been presumed that numerous species from diverse taxa benefit from its hypometabolic effect. To investigate the phylogenetic prevalence of urea-induced hypometabolism, we studied four species of urea accumulators from the clades Amphibia (Spea bombifrons and Ambystoma tigrinum), Reptilia (Malaclemys terrapin), and Gastropoda (Anguispira alternata), and one amphibian species (R. pipiens) that does not accumulate urea during dormancy. We measured rates of oxygen consumption (VO(2)) of excised organ samples from dormant animals in the presence or absence of physiological concentrations of urea. Three of the four urea-accumulating species had at least one organ whose VO(2) was significantly decreased by urea treatment. However, VO(2) of organs from R. pipiens, the one species tested that does not accumulate urea during dormancy, was not affected by urea treatment. Our results support the hypothesis that urea accumulation can reduce metabolic rate of dormant animals and provide a base for further investigation into the evolution of urea-induced hypometabolism., ((c) 2009 Wiley-Liss, Inc.)

The basilar papilla (BP) in the frog inner ear is a relatively simple auditory receptor. Its hair cells are embedded in a stiff support structure, with the stereovilli connecting to a flexible tectorial membrane (TM). Acoustic energy passing the papilla presumably causes displacement of the TM, which in turn deflects the stereovilli and stimulates the hair cells. Auditory neurons that contact the BP's hair cells are known to have nearly identical characteristic frequencies and frequency selectivity. In this paper, we present optical measurements of the mechanical response of the TM. Results were obtained from five specimens. The TM displacement was essentially in phase across the membrane, with the largest amplitudes occurring near the hair cells. The response was tuned to a frequency near 2 kHz. The phase accumulated over at least 270 degrees across the measured frequencies. The tuning quality Q(10dB) values were calculated; the average Q(10dB) was 2.0 +/- 0.8 (standard deviation). Our results are comparable to those of neural-tuning curves in the same and a similar species. Also, they are in agreement with the response of an associated structure-the contact membrane-in a closely related species. Our data provides evidence for a mechanical basis for the frequency selectivity of the frog's BP.

Arsenic (As) is mainly released to the environment from anthropogenic sources, with inorganic pentavalent As (As [V]) predominant in surface water. In this study, Rana pipiens were exposed to As (V) in water at environmentally relevant concentrations (control, 10, 20, 150, 500, and 1,000 microg l(-1)) in a static-renewal system from post-hatch stage through metamorphosis for 113 days. There was no significant effect of As exposure on tadpole survival, growth, and percent metamorphosis. Maximum swimming speed was significantly slower in the As-treated groups compared with the control. During the period of tail resorption (Gosner stage 42-46), no significant differences in age at metamorphosis, survival, length of tail resorption period, snout-vent length of metamorphs, and sex ratio were found among treatments. Whole body As concentrations ranged from <0.6 to 5.31 mg kg(-1) dry mass, and were significantly higher in the 150, 500, and 1,000 microg l(-1) treatments than the control. Based on our data, larval tissue concentrations of As close to that reported in previous field studies were not associated with any significant effects except decreased tadpole swimming speed.

This study presents a direct comparison of the ligand binding and signaling profiles of a mammalian and non-mammalian mu opioid receptor. Opioid ligand binding and agonist potencies were determined for an amphibian (Rana pipiens) mu opioid receptor (rpMOR) and the human mu opioid receptor (hMOR) in transfected, intact Chinese hamster ovary (CHO) cells. Identical conditions were employed such that statistically meaningful differences between the two receptors could be determined. Identifying these differences is an important first step in understanding how evolutionary changes affect ligand binding and signaling in vertebrate opioid receptors. As expected, the rank of opioid ligand affinity for rpMOR and hMOR was consistent with the ligands' previously characterized type-selectivity. However, most of the opioid ligands tested had significant differences in affinity for rpMOR and hMOR. For example, the mu-selective agonist, DAMGO ([d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin), had a 10.9-fold greater affinity (K(i)) for hMOR (K(i)=268 nM) than rpMOR (K(i)=2914 nM). In addition, differences in signaling between these receptors were found by measuring inhibition of cAMP accumulation by morphine or DAMGO. DAMGO was significantly more potent (13.6-fold) in CHO cells expressing hMOR versus those expressing rpMOR. In addition, a significantly greater maximal inhibition was elicited by both opioid agonists in cells expressing hMOR. In summary, this study supports an ongoing effort to better understand how vertebrate evolution has shaped opioid receptor properties and function.

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

We find that completion of the first meiotic division in Rana pipiens oocytes requires the sequential action of at least two steroids: progesterone and one or more subsequent polar metabolites of progesterone. Progesterone binding in vitro to oocyte surface receptors increases during the first 4-5h of exposure, followed by internalization of plasma membrane together with membrane-bound progesterone over the next hour. The internalized progesterone is metabolized to highly polar polyhydroxylated steroid(s) prior to nuclear membrane disappearance at 8-9h. Polar steroids alone cannot induce meiosis, but do so in oocytes pretreated with progesterone for 1h. Similarly, the non-metabolizable progestin R5020 cannot induce meiosis but does if oocytes are subsequently exposed to polar steroids. An inhibitor of steroid alpha-reductase (4-MA) prevents both progesterone metabolism and progesterone-induced meiosis. However, meiosis does occur if 4-MA is followed by a polar steroid. Thus, progesterone binding at the oocyte surface initiates a process which requires polar progesterone metabolites for completion of the first meiotic division.

Chloride conductance across the isolated skin of Rana pipiens shows a voltage-activated component (G(Cl)(V)) which requires the presence of mucosal Cl. G(Cl)(V) is normally low or dormant. It is stimulated by elevated intracellular cAMP, irrespective whether originating from application of ss-adrenergic agonists (isoproterenol), stimulators of the adenylyl-cyclase (forskolin), inhibitors of the phosphodiesterases (isobutyl-methyl-xanthine) or membrane-permeable cAMP analogues (CPT-cAMP). Baseline G(Cl) under inactivating conditions increases also with cAMP dose-dependently. The data indicate that cAMP is a central regulator of the passive, conductive chloride transport across amphibian skin.