Amphibian Physiology Research Articles

Current State of Conservation Physiology for Amphibians: Major Research Topics and Physiological Parameters.

Analysis of physiological responses can be used to assess population health, identify threat factors, and understand mechanisms of stress. In addition to this, conservation physiologists have sought to establish potential management strategies for environmental change and evaluate the effectiveness of conservation efforts. From past to present, the field of conservation physiology is developing in an increasingly broader context. In this review, we aim to categorize the topics covered in conservation physiology research on amphibians and present the measured physiological parameters to provide directions for future research on conservation physiology. Physiological responses of amphibians to environmental stressors are the most studied topic, but conservation physiological studies on metamorphosis, habitat loss and fragmentation, climate change, and conservation methods are relatively lacking. A number of physiological indices have been extracted to study amphibian conservation physiology, and the indices have varying strengths of correlation with each subject. Future research directions are suggested to develop a comprehensive monitoring method for amphibians, identify interactions among various stressors, establish physiological mechanisms for environmental factors, and quantify the effects of conservation activities on amphibian physiology.

The Adaptive Host Manipulation Hypothesis: Parasites Modify the Behaviour, Morphology, and Physiology of Amphibians

Parasites have evolved different strategies to increase their transmission from one host to another. The Adaptive Host Manipulation hypothesis states that parasites induce modifications of host phenotypes that could maximise parasite fitness. There are numerous examples of parasite manipulation across a wide range of host and parasite taxa. However, the number of studies exploring the manipulative effects of parasites on amphibians is still scarce. Herein, we extensively review the current knowledge on phenotypic alterations in amphibians following parasite infection. Outcomes from different studies show that parasites may manipulate amphibian behaviours to favour their transmission among conspecifics or to enhance the predation of infected amphibians by a suitable definite host. In addition, parasites also modify the limb morphology and impair locomotor activity of infected toads, frogs, and salamanders, hence facilitating their ingestion by a final host and completing the parasite life cycle. Additionally, parasites may alter host physiology to enhance pathogen proliferation, survival, and transmission. We examined the intrinsic (hosts traits) and extrinsic (natural and anthropogenic events) factors that may determine the outcome of infection, where human-induced changes of environmental conditions are the most harmful stressors that enhance amphibian exposure and susceptibility to parasites.

Biochemical and osmoregulatory responses of the African clawed frog experimentally exposed to salt and pesticide

Salinization and pollution are two main environmental stressors leading deterioration to water quality and degradation of aquatic ecosystems. Amphibians are a highly sensitive group of vertebrates to environmental disturbance of aquatic ecosystems. However, studies on the combined effect of salinization and pollution on the physiology of amphibians are limited. In this study, we measured the standard metabolic rate (SMR) and biochemical parameters of adult males of the invasive frog Xenopus laevis after 45 days of exposure to contrasting salinity environments (400 and 150 mOsm NaCl) with either 1.0 μg/L of the organophosphate pesticide chlorpyrifos (CPF) or pesticide-free medium. Our results revealed a decrease in SMR of animals exposed to the pesticide and in the ability to concentrate the plasma in animals exposed simultaneously to both stressors. The lack of ability to increase plasma concentration in animals exposed to both salt water and CPF, suggests that osmoregulatory response is decreased by pesticide exposure. In addition, we found an increase of liver citrate synthase activity in response to salt stress. Likewise, the liver acetylcholinesterase (AChE) activity decreased by 50% in frogs exposed to salt water and CPF and 40% in those exposed only to CPF, which suggest an additive effect of salinity on inhibition of AChE. Finally, oxidative stress increased as shown by the higher lipid peroxidation and concentration of aqueous peroxides found in the group exposed to salt water and pesticide. Thus, our results revealed that X. laevis physiology is compromised by salinization and pesticide exposure to both environmental stressors join.

Time Course of Splenic Cytokine mRNA and Hormones during a Lipopolysaccharide-Induced Inflammation in Toads.

Inflammation comprises alterations in glucocorticoids (in amphibians, corticosterone-CORT) and melatonin (MEL) levels, two hormones with immunomodulatory effects on cytokine production in several vertebrates. Cytokines mediate inflammation progress differently depending on their function. While some are secreted during the acute phase of the immune response, others prevail during the resolution phase. Major efforts have been made to understand the interaction of endocrine mediators and cytokine production in endotherms, but little is known for ectotherms so far. Characterizing the stages of inflammation and their interplay with endocrine mediators is crucial for an assertive and integrative approach to amphibian physiology and ecoimmunology. Herein, we investigated CORT and MEL plasma levels as well as splenic cytokine (IL-1β, IL-6, and IL-10) mRNA levels during the progression of the inflammatory response in toads (Rhinella diptycha) in four time-points (1, 3, 6, and 18 h) after an immune challenge with lipopolysaccharide (LPS) using independent samples. Toads were responsive to LPS, with all hormones and cytokines affected by LPS. IL-1β and IL-6 were up-regulated after 1 h, but IL-1β decreased right after 3 h, while IL-6 sustained up-regulation throughout all time-points. IL-10 had not been detected until 6 h post-LPS-stimulation, when it showed up-regulation, along with a CORT increase at the same time-point. After 18 h, CORT levels were still high, and IL-1β was up-regulated again, along with up-regulated IL-6 and an IL-10 decrease. We also found positive correlations between IL-1β with IL-6 for LPS and saline groups. LPS-treated individuals showed an overall decrease in MEL plasma levels compared to saline counterparts. Our results showcase the early endocrine and molecular events of the amphibian immune response. We also report activation of the hypothalamus-pituitary-interrenal (HPI) axis during inflammation and increasing evidence for an immune-pineal axis to be described in amphibians.

Realistic exposure to fipronil, 2,4-D, vinasse and their mixtures impair larval amphibian physiology

Expansion of sugarcane crops may have contributed to the increased contamination of native habitats in Brazil. Several species of amphibians inhabit ponds formed in flooded farmlands, where pesticide concentrations are usually high. This study evaluated the ecotoxicological effects of the sugarcane pesticides fipronil and 2,4-D, as well as the fertilizer vinasse (isolated and mixed), on physiological responses of Leptodactylus fuscus and Lithobates catesbeianus tadpoles. In situ assays were conducted in mesocosms with concentrations based on the doses recommended by the manufacturer. Vinasse (1.3% dilution) caused 100% tadpoles’ mortality immediately after its application. Fipronil and/or 2,4-D altered antioxidant and biotransformation responses, induced neurotoxicity and changed lipid contents in tadpoles. A multivariate approach indicated that the mixture of pesticides induced most of the sublethal effects in both tadpole species, in addition to the isolated fipronil in L. fuscus. Fipronil alone increased glucose-6-phosphate dehydrogenase (G6PDH) activity, decreased acetylcholinesterase (AChE) and total lipid contents, and altered some individual lipid classes (e.g., free fatty acids and acetone-mobile polar lipids) in L. fuscus. The interaction between fipronil and 2,4-D in this species were more evident for lipid contents, although enzymatic alterations in G6PDH, AChE and glutathione-S-transferase (GST) were also observed. In L. catesbeianus, the mixture of pesticides reduced triglycerides and total lipids, as well as increased GST and decreased AChE activities. The detoxifying enzyme carboxylesterase was reduced by 2,4-D (alone or in mixture) in both species. Isolated pesticides also modulated specific lipid classes, suggesting their disruptive action on energy metabolism of tadpoles. Our study showed that fipronil, 2,4-D, and vinasse, individually or mixed, can be harmful to amphibians during their larval phase, causing mortality or impairing their functional responses.

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

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

Shifts in sensitivity of amphibian metamorphosis to endocrine disruption: the common frog (Rana temporaria) as a case study.

Effective conservation actions require knowledge on the sensitivity of species to pollution and other anthropogenic stressors. Many of these stressors are endocrine disruptors (EDs) that can impair the hypothalamus-pituitary-thyroid axis and thus alter thyroid hormone (TH) levels with physiological consequences to wildlife. Due to their specific habitat requirements, amphibians are often sentinels of environmental degradation. We investigated how altered TH levels affected the bioenergetics of growth and development (i.e. age, size, metabolism, cardiac function and energy stores) before, during and after metamorphosis in the European common frog (Rana temporaria). We also determined how ontogenetic stage affected susceptibility to endocrine disruption and estimated juvenile performance. TH levels significantly affected growth and energetics at all developmental stages. Tadpoles and froglets exposed to high TH levels were significantly younger, smaller and lighter at all stages compared to those in control and low TH groups, indicating increased developmental and reduced growth rates. Across all ontogenetic stages tested, physiological consequences were rapidly observed after exposure to EDs. High TH increased heart rate by an average of 86% and reduced energy stores (fat content) by 33% compared to controls. Effects of exposure were smallest after the completion of metamorphosis. Our results demonstrate that both morphological and physiological traits of the European common frog are strongly impacted by endocrine disruption and that ontogenetic stage modulates the sensitivity of this species to endocrine disruption. Since endocrine disruption during metamorphosis can impair the physiological stress response in later life stages, long-term studies examining carry-over effects will be an important contribution to the conservation physiology of amphibians.

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.

Non-invasive Methods for Measuring and Monitoring Stress Physiology in Imperiled Amphibians

Global climate change is negatively impacting global biodiversity and ectothermic vertebrates, with amphibians being the most imperiled vertebrate taxa. Increased mean global atmospheric temperatures, high rates of habitat degradation, and exposure to infectious diseases, such as chytridiomycosis, have contributed to population declines and extinctions of rare and endangered amphibian species. Field-based monitoring of physiological endocrine traits can help determine the sub-lethal effects of environmental stressors and provide early alerts when populations are chronically stressed. Recent advances in amphibian stress endocrinology include the development and use of non-invasive methods to quantify the glucocorticoid, or stress biomarker, corticosterone. Non-invasive methods, such as urinary, skin and buccal swabs, and water-borne hormone monitoring methods (suited for terrestrial and aquatic dwelling species), are available to quantify baseline and short-term physiological stress responses of amphibians under field settings. In this review, we illustrate, by using two case studies of aquatic and terrestrial amphibian species, the applications of non-invasive corticosterone monitoring methods to advance the ecological knowledge and conservation of imperiled amphibian species, discuss the limitations of these methods, and provide future directions for the use of non-invasive hormone monitoring methods. We highlight the use of non-invasive field endocrinology methods to monitor the impacts of environmental stressors on the physiology of amphibians, which can be applied to advance ecological research, conservation, and management of imperiled species.

Innate immunity and antioxidant costs of low temperatures in native green treefrogs (Hyla cinerea) and invasive tropical Cuban treefrogs (Osteopilus septentrionalis)

Thermal tolerance plays a role in the geographic range over which different species can survive, and temperature fluctuations can affect amphibian physiology. We studied physiological costs associated with low temperatures in green treefrogs (Hyla cinerea), which are native to the United States, and tropical, non-native Cuban treefrogs (Osteopilus septentrionalis) that have invaded Florida. Specifically, we examined variation in innate immunity and skin antioxidant capacity. To complete the study, 107 treefrog tadpoles were raised through metamorphosis and, as young frogs, divided into a control group maintained at room temperature of approximately 24°C and an experimental group at approximately 8°C. Individuals in the low temperature group were placed in the cold temperatures for 6-8 hours per day for 14 days to simulate early fall nocturnal ambient temperatures in northern regions of states along the Gulf of Mexico in the United States. Cuban treefrogs had a significantly greater survival rate than green treefrogs in the control group, but there was no significant difference in survival between experimental groups. We also found that each species was negatively affected by low temperatures, with a decrease in bacterial killing ability and antioxidants on their skin, but the Cuban treefrogs were affected to a much greater extent. These findings suggest that the spread of Cuban treefrogs into areas north of their current invasive range may be slowed by physiological costs of low temperatures, but we do not expect significantly greater mortality rates associated with cold alone than treefrogs already occupying such a range.

Thermal equilibrium and temperature differences among body regions in European plethodontid salamanders

Information on species thermal physiology is extremely important to understand species responses to environmental heterogeneity and changes. Thermography is an emerging technology that allows high resolution and accurate measurement of body temperature, but until now it has not been used to study thermal physiology of amphibians in the wild. Hydromantes terrestrial salamanders are strongly depending on ambient temperature for their activity and gas exchanges, but information on their body temperature is extremely limited. In this study we tested if Hydromantes salamanders are thermoconform, we assessed whether there are temperature differences among body regions, and evaluated the time required to reach the thermal equilibrium. During summers of 2014 and 2015 we analysed 56 salamanders (Hydromantes ambrosii and Hydromantes italicus) using infrared thermocamera. We photographed salamanders at the moment in which we found them and 1, 2, 3, 4, 5 and 15min after having kept them in the hands. Body temperature was equal to air temperature; salamanders attained the equilibrium with air temperature in about 8min, the time required to reach equilibrium was longer in individuals with large body size. We detected small temperature differences between body parts, the head being slightly warmer than the body and the tail (mean difference: 0.05°C). These salamanders quickly reach the equilibrium with the environment, thus microhabitat measurement allows obtaining accurate information on their tolerance limits.

Usefulness of Animal Simulator Software in teaching Amphibian Physiology Practical for 1st Prof MBBS Students

Background & Objectives: Amphibian experiments in Physiology Practical classes are at present taught by using simple lecture-demonstrations as killing of animals for laboratory use has become controversial especially after the intervention of animal rights activists. Animal Simulation method could be used as an alternative to it.The present study tried to see the knowledge gained by the 1st Prof MBBS students by using Animal Simulation model in Amphibian Physiology Practical classes as compared to conventional Lecture-Demonstration method. It also tried to find out that the difference in knowledge gain by Poor performing students as compared to Good Performers. The student’s Perception on this simulation based T-L Methodology was also surveyed. Material & Methods: This interventional, prospective study was done in the department of Physiology, B. S. Medical College, Bankura, after obtaining proper written approval from the institutional ethics committee. Pre-test was conducted by a validated Questionnaire consisting 25 MCQs. Then the class of 150 1st Year medical students was divided into two groups of 75 students each by Systematic Random Sampling. Group 1 or Case attended Animal Simulation ((Reed Elsevier India Pvt. Ltd, product@animalsimulator.com) Method while other batch of students (Group 2, Control) attended Lecture-demonstration. After 15 days both the groups were tested with the same questionnaire (Post Test). Pre-Test & Post-Test scores were analyzed using SPSS statistical software. Group 1 student’s perception to the simulation based teaching was surveyed by a questionnaire. Cross over was done after completion of the study. Result: The new method of Simulation based teaching of nerve-muscle Physiology of 1st Prof MBBS students was found more effective than the Traditional Lecture- Demonstration method. This new method is particularly helpful for slow learners. Students also found it interesting, student centric and useful. Conclusion: Simulation based teaching of Amphibian Physiology is a better method that needs to be implemented on a wider scale in 1st Prof MBBS Physiology curriculum.

Acute Thermal Stressor Increases Glucocorticoid Response but Minimizes Testosterone and Locomotor Performance in the Cane Toad (Rhinella marina)

Climatic warming is a global problem and acute thermal stressor in particular could be considered as a major stressor for wildlife. Cane toads (Rhinella marina) have expanded their range into warmer regions of Australia and they provide a suitable model species to study the sub-lethal impacts of thermal stressor on the endocrine physiology of amphibians. Presently, there is no information to show that exposure to an acute thermal stressor could initiate a physiological stress (glucocorticoid) response and secondly, the possible effects on reproductive hormones and performance. Answering these questions is important for understanding the impacts of extreme temperature on amphibians. In this study, we experimented on cane toads from Queensland, Australia by acclimating them to mildly warm temperature (25°C) and then exposing to acute temperature treatments of 30°, 35° or 40°C (hypothetical acute thermal stressors). We measured acute changes in the stress hormone corticosterone and the reproductive hormone testosterone using standard capture and handling protocol and quantified the metabolites of both hormones non-invasively using urinary enzyme-immunoassays. Furthermore, we measured performance trait (i.e. righting response score) in the control acclimated and the three treatment groups. Corticosterone stress responses increased in all toads during exposure to an acute thermal stressor. Furthermore, exposure to a thermal stressor also decreased testosterone levels in all toads. The duration of the righting response (seconds) was longer for toads that were exposed to 40°C than to 30°, 35° or 25°C. The increased corticosterone stress response with increased intensity of the acute thermal stressor suggests that the toads perceived this treatment as a stressor. Furthermore, the results also highlight a potential trade-off with performance and reproductive hormones. Ultimately, exposure acute thermal stressors due to climatic variability could impact amphibians at multiple eco-physiological levels through impacts on endocrine physiology, performance and potentially fitness traits (e.g. reproductive output).

A non-invasive water-borne hormone assay for amphibians

Anthropogenic disturbances have been implicated in the rapid decline of amphibians. Disturbances, such as disease and poor water quality, might cause changes in the physiology of amphibians resulting in chronic stress, which can result in decreased growth and development as well as immunosuppression. In amphibians, corticosterone (CORT) is the main hormone released in response to stressors. We took the first steps towards validating a new, non-invasive, technique to assay CORT in amphibians using a water-borne collection method previously used only with fish. In validation of this technique, we found a significant positive correlation between release rates of water-borne CORT and levels of CORT in circulating plasma in adults of the San Marcos salamander, Eurycea nana, and the common midwife toad, Alytes obstetricans. These results indicate that water-borne CORT can be used as a proxy for plasma CORT. Additionally, we examined basic background information on the physiological states of these two species. We found that captive-reared salamanders had significantly lower release rates of CORT than field-collected salamanders. Field-collected salamanders had significantly higher CORT release rates 24 h after capture and transfer to the laboratory. For tadpoles, we found that field-collected tadpoles did not have significantly different CORT release rates than those maintained in the laboratory for four months. Our research indicates that this method of water-borne hormone collection should be viable for many species of amphibians; however, further validation via adrenocorticotropic hormone (ACTH) challenges is required. This method can be a useful tool for assessing the physiological state of laboratory and field populations of amphibians and the effects of urbanization, pesticides and diseases. An important benefit of this method is that it allows for repeated measures of the same individuals and can be less stressful than drawing blood.

Exploring nervous system transcriptomes during embryogenesis and metamorphosis in Xenopus tropicalis using EST analysis

BackgroundThe western African clawed frog Xenopus tropicalis is an anuran amphibian species now used as model in vertebrate comparative genomics. It provides the same advantages as Xenopus laevis but is diploid and has a smaller genome of 1.7 Gbp. Therefore X. tropicalis is more amenable to systematic transcriptome surveys. We initiated a large-scale partial cDNA sequencing project to provide a functional genomics resource on genes expressed in the nervous system during early embryogenesis and metamorphosis in X. tropicalis.ResultsA gene index was defined and analysed after the collection of over 48,785 high quality sequences. These partial cDNA sequences were obtained from an embryonic head and retina library (30,272 sequences) and from a metamorphic brain and spinal cord library (27,602 sequences). These ESTs are estimated to represent 9,693 transcripts derived from an estimated 6,000 genes. Comparison of these cDNA sequences with protein databases indicates that 46% contain their start codon. Further annotation included Gene Ontology functional classification, InterPro domain analysis, alternative splicing and non-coding RNA identification. Gene expression profiles were derived from EST counts and used to define transcripts specific to metamorphic stages of development. Moreover, these ESTs allowed identification of a set of 225 polymorphic microsatellites that can be used as genetic markers.ConclusionThese cDNA sequences permit in silico cloning of numerous genes and will facilitate studies aimed at deciphering the roles of cognate genes expressed in the nervous system during neural development and metamorphosis. The genomic resources developed to study X. tropicalis biology will accelerate exploration of amphibian physiology and genetics. In particular, the model will facilitate analysis of key questions related to anuran embryogenesis and metamorphosis and its associated regulatory processes.

Corticosterone concentrations in free-living spotted salamanders ( Ambystoma maculatum)

A non-lethal technique for drawing repeated blood samples from spotted salamanders ( Ambystoma maculatum) was used to examine sex, seasonal, and capture technique differences in the physiological stress response under natural, undisturbed conditions. Baseline and stress-induced (30 min of handling and restraint) corticosterone (CORT) concentrations were measured at night during the spring migrations into and out of the breeding pond, as well as during the fall migration to over-wintering sites. Females had significantly higher CORT concentrations than males during the spring migration toward the breeding pond, but this difference was not present when animals emerged from the pond post-breeding or during the fall migration. CORT concentrations did not vary seasonally, but during the acute stress response, CORT concentrations nearly doubled during the spring inbound migration, exhibited a nearly significant increase during the spring outbound migration, and did not change in the fall. Allowing animals to sit overnight in the buckets in which they were captured elicited CORT concentrations that tended to be higher (although not statistically significant) than when animals were sampled on the night of capture. The ability to sample blood from spotted salamanders using a non-lethal technique offers an opportunity to better understand both amphibian physiology and ecology.

Benzoate X Receptors α and β Are Pharmacologically Distinct and Do Not Function as Xenobiotic Receptors

The Xenopus benzoate nuclear hormone receptors, BXRalpha and BXRbeta, share 82% identity within their ligand-binding domains and are classified as members of the NR1I2 subfamily that includes the mammalian steroid and xenobiotic receptor, SXR/PXR. Although alkyl benzoates have been identified as endogenous ligands, the exact role of the benzoate receptors in amphibian physiology has not been established. In this report, we show that BXRalpha and BXRbeta are pharmacologically distinct from each other: BXRalpha is more promiscuous than BXRbeta with respect to both ligand specificity and co-activator recruitment. BXRalpha can be transactivated by a number of benzoate derivatives including 4-amino-butylbenzoate (4-ABB), 4-hydroxy-butylbenzoate (4-HBB), 3-hydroxy ethyl benzoate (3-HEB), and benzyl benzoate, but only 4-HBB acts as an agonist for both receptors. Furthermore, BXRalpha-specific agonists such as 4-ABB, chlorpyrifos, and trifluralin act as antagonists on BXRbeta. BXRs are widely distributed in adult tissues but do not show any enrichment in liver and intestine, major sites of SXR/PXR expression that are critical in xenobiotic metabolism. Neither BXR shows the broad specificity toward steroids or xenobiotics exhibited by SXR/PXR. Therefore, we conclude that the BXRs are pharmacologically distinct from each other and unlikely to serve as xenobiotic sensors.

200 YEARS OF AMPHIBIAN WATER ECONOMY: FROM ROBERT TOWNSON TO THE PRESENT

ABSTRACTIn the 1790s, Robert Townson established the main features of the water economy of terrestrial amphibians: rapid evaporative water loss in dry surroundings,‘drinking’ by absorption of water through the abdominal skin pressed against moist substrates, and use of the urinary bladder as a reservoir from which water is reabsorbed on land. This knowledge was of little interest to the establishment in the first half of the nineteenth century of experimental physiology as a basic medical discipline, when frogs became models in the elucidation of general physiological processes. Townson's pioneer contributions to amphibian physiology were forgotten for 200 years (Jørgensen 1994 b). Durig (1901) and particularly Overton (1904) restored knowledge about amphibian water economy to the level reached by Townson, but the papers had little impact on the young science of animal physiology because they primarily aimed at elucidating the transport of fluids across membranes. Frog skin remained a model preparation in such studies throughout the century. With the establishment of terrestrial ecology early in the century, the relations of animals, including amphibians, to water became a central theme. Concurrently with comparative studies of amphibian water economy in an ecological setting, the subject proceeded as an aspect of animal osmoregulation. Adolph (1920‐1930) and Rey (1937 a) established the highly dynamic nature of water balance in amphibians in water and on land. Their observations indicated functional links between environment, skin and kidneys, the nature of which remained to be explored. Thorson & Svihla (1943) reopened the ecological approach in a comparative study of the relations between amphibian habitat and tolerance of dehydration. By mid‐century, the central themes of amphibian adaptations to terrestrial modes of life were re‐established, except for the function of the bladder as a water‐depot. During the following decades, a rich literature appeared, particularly focusing on adaptations of amphibians to arid environments. Thus, in the 1970s, it was found that ‘waterproofing’ of the highly permeable skins by means of skin secretions had evolved independently in several families of tropical arboreal frogs, and that a number of amphibians that aestivate whilst burrowed in dry soil could reduce evaporation by forming cocoons from shed strata cornea. In 1950–1970 the role of bladder urine as a water depot in terrestrial amphibians was recognized: this did not change the established view of water balance in terrestrial amphibians as alternating between dehydration on land and rehydration in response to the deficit in body water. Amphibians may, however, maintain normal water balance whether the ambient medium is water or air by means of little understood integrated mechanisms in control of cutaneous drinking behaviour, water permeability of the skin and bladder wall, and urine production.

200 years of amphibian water economy: from Robert Townson to the present.

In the 1790s, Robert Townson established the main features of the water economy of terrestrial amphibians: rapid evaporative water loss in dry surroundings, 'drinking' by absorption of water through the abdominal skin pressed against moist substrates, and use of the urinary bladder as a reservoir from which water is reabsorbed on land. This knowledge was of little interest to the establishment in the first half of the nineteenth century of experimental physiology as a basic medical discipline, when frogs became models in the elucidation of general physiological processes. Townson's pioneer contributions to amphibian physiology were forgotten for 200 years (Jørgensen 1994b). During (1901) and particularly Overton (1904) restored knowledge about amphibian water economy to the level reached by Townson, but the papers had little impact on the young science of animal physiology because they primarily aimed at elucidating the transport of fluids across membranes. Frog skin remained a model preparation in such studies throughout the century. With the establishment of terrestrial ecology early in the century, the relations of animals, including amphibians, to water became a central theme. Concurrently with comparative studies of amphibian water economy in an ecological setting, the subject proceeded as an aspect of animal osmoregulation. Adolph (1920-1930) and Rey (1937a) established the highly dynamic nature of water balance in amphibians in water and on land. Their observations indicated functional links between environment, skin and kidneys, the nature of which remained to be explored. Thorson & Svihla (1943) reopened the ecological approach in a comparative study of the relations between amphibian habitat and tolerance of dehydration. By mid-century, the central themes of amphibian adaptations to terrestrial modes of life were re-established, except for the function of the bladder as a water-depot. During the following decades, a rich literature appeared, particularly focusing on adaptations of amphibians to arid environments. Thus, in the 1970s, it was found that 'waterproofing' of the highly permeable skins by means of skin secretions had evolved independently in several families of tropical arboreal frogs, and that a number of amphibians that aestivate whilst burrowed in dry soil could reduce evaporation by forming cocoons from shed strata cornea. In 1950-1970 the role of bladder urine as a water depot in terrestrial amphibians was recognized: this did not change the established view of water balance in terrestrial amphibians as alternating between dehydration on land and rehydration in response to the deficit in body water. Amphibians may, however, maintain normal water balance whether the ambient medium is water or air by means of little understood integrated mechanisms in control of cutaneous drinking behaviour, water permeability of the skin and bladder wall, and urine production.

Robert Townson's observations on amphibian water economy revived

The neglected works of Robert Townson (1799) anticipated the establishment of the basic features in the physiology of amphibian water economy by about a century and a half. A re-examination of Townson's published journals on water balance in tree frogs ( Hyla arborea) acclimated to a simulated terrestrial habitat with free access to water substantially widened the scope of the work. The tree frogs, and other terrestrial anurans, did not void urine on land, but the water was reabsorbed from the bladder to substitute water lost by evaporation. The tree frogs mostly took in water by absorption through the abdominal skin (cutaneous drinking) before they became dehydrated, indicating that drinking was anticipatory. After a drinking episode, water was stored in the bladder in amounts corresponding to 25–50% of the body mass. Townson's pioneering contributions to the adaptational physiology of amphibians were disregarded by contemporary animal physiology, which basically served to elucidate functions in higher animals and ultimately humans.