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.     

Water retention and plasma and urine composition in toads (Bufo viridis Laur.) under burrowing conditions

Summary Osmoregulation in the terrestrial toad,Bufo viridis, was studied under burrowing conditions in the laboratory. The toads can live for over 3 months burrowed in soil containing 9–10% moisture, maintaining constant body volume due to a large increase in the plasma osmolality, contributed mainly by urea. Water content of the tissues remains constant. Relatively large volumes of urine are stored in the urinary bladder during water restriction. The osmolality of the urine does not exceed that of the plasma. Urea uptake across the skin was measured in vitro and was greatly elevated in skins from the burrowed toads. The increase in plasma osmolality enables greater water absorption from the soil under water restricted conditions while the water content of the tissues is maintained constant since cell membranes are highly permeable to urea. It is concluded that the urea accumulating ability and urea tolerance form the basis for both the terrestriality and salt adaptability of this and other amphibian species.     

Behavioral, molecular and integrative mechanisms of amphibian osmoregulation

Amphibian water balance has been studied at many levels of biological order. Terrestrial species must react to environmental cues that relate to water availability while some arboreal species have cutaneous skin secretions that can reduce evaporative water loss. The Indian tree frog. Polypedates maculatus, uses cutaneous secretions and wiping behavior to lower evaporation but also relies on moist microclimates to endure prolonged survival away from water. The related species, P. leucomystax, inhabits wetter forest habitats. Preliminary studies with this species are unable to demonstrate the expression of wiping behavior, indicating that arid habitats may be a powerful selective force for this behavior. Laboratory experiments on rehydrating toads in the genus Bufo indicate that animals are able to detect changes in barometric pressure and humidity that might result in the availability of water under field situations. Experiments with Bufonid species and with spadefoot toads, Scaphiopus couchi, show that the peptide hormone, angiotensin II, stimulates cutaneous drinking in a similar manner seen for oral drinking by other vertebrate classes. Amphibian tissues have long been used as a model for the study of basic physiological principles of epithelial ion and water transport. Recent progress with tissue cultures has provided information on the molecular structure of ion and water channels that can be applied to obtain a better understanding, at the molecular level, of ion and water balance strategies used by the wide variety of amphibian species. Terrestrial amphibians are more tolerant of dehydration than are other vertebrates and are able to store dilute urine in their urinary bladder. Toads appear to be able to detect the presence of water in their bladders in addition to the availability of water in their environment. Dehydrated toads are able to rehydrate very rapidly by the coordination of behavioral and physiological mechanisms to enhance cutaneous water absorption. The integration of behavior with cutaneous water gain, renal handling of ions and water and the role of the lymphatic system in overall water balance involves complex interactions between neural and hormonal factors. Experiments are summarized that describe the contribution of individual factors however much more information is needed before the nature of these interactions are fully understood.     

Effects of dehydration on plasma osmolality, thirst-related behavior, and plasma and brain angiotensin concentrations in Couch's spadefoot toad, Scaphiopus couchii

Under dehydrating conditions, many terrestrial vertebrates species exhibit increases in plasma osmolality and their drinking behavior. Under some circumstances, this behavioral change is accompanied by changes in plasma and central angiotensin concentrations, and it has been proposed that these changes in angiotensin levels induce the thirst-related behaviors. In response to dehydration, the spadefoot toad, Scaphiopus couchii, exhibits thirst-related behavior in the form of cutaneous drinking. This behavior has been termed water absorption response (WR) behavior. Spadefoot toads live in harsh desert environments and are subject annually to dehydrating conditions that may induce thirst-related behavior. We tested the hypothesis that an increase in WR behavior is associated with both an increase in plasma osmolality and an increase in plasma and brain angiotensin concentrations. First, we determined the degree of dehydration that was necessary to initiate WR behavior. Animals dehydrated to 85% of their standard bladder-empty weight via deprivation of water exhibited WR behavior more frequently than control toads left in home containers with water available. Next, using the same dehydration methods, we determined the plasma osmolality and sodium concentrations of dehydrated toads. Toads dehydrated to 85% standard weight also had a significant increase in plasma osmolality, but exhibited no overall change in plasma sodium concentrations, indicating that while an overall increase in plasma osmolality appears to be associated with WR behavior in S. couchii, changes in sodium concentrations alone are not sufficient to induce the behavior. Finally, plasma and brain angiotensin concentrations were measured in control toads and toads dehydrated to 85% standard weight. Plasma and brain angiotensin concentrations did not increase in dehydrated toads, indicating that dehydration-induced WR behavior that is associated with changes in plasma osmolality may not be induced by changes in endogenous angiotensin concentrations in S. couchii.     

Comparison of dehydration and angiotensin II-stimulated cutaneous drinking in toads, Bufo punctatus

Toads (Bufo punctatus) use a sequence of two postures to place the ventral skin on a moist surface and absorb water osmotically. First, the skin contacts the surface (seat patch down, SPD), and then the hindlimbs are abducted to maximize skin contact area (water absorption response, WR). Toads modulated behavior in response to hydration status and osmotic content of the hydration source. Dehydrated toads placed on water displayed both SPD and WR. Hydrated toads injected with angiotensin II (AII) displayed SPD longer than Ringer-injected controls but did not initiate WR and absorbed less water than dehydrated toads. These results suggest that dehydration has a more robust dipsogenic effect than AII. Dehydrated toads placed on 250 mM NaCl briefly initiated SPD but not WR. The addition of amiloride to the hyperosmotic salt solution resulted in brief display of WR but no water loss. Hydrated toads placed on 250 mM NaCl showed shorter periods of SPD behavior. The combination of AII injection and amiloride addition to the salt solution increased SPD initiation but SPD duration was short and water loss was prevented. Neither AII nor dehydration overrides chemosensory mechanisms in the skin that suppress cutaneous drinking from hypertonic solutions.     

Behavioural thermoregulation of the Andean toad (Bufo spinulosus) at high altitudes

Summary The body temperature of free-ranging Andean toadsBufo spinulosus was measured either directly or radiotelemetrically during two 15-day periods at 3200 m elevation in the Mantaro Valley, Central Perú. All toads attempted to maintain their diurnal sum of body temperature within a narrow range. Consequently thermoregulatory behaviour differed according to cloud cover and precipitation. If the sky was clear, toads emerged from their hiding place and exposed themselves to solar radiation during 3–5 h in the morning. Core temperature increased up to 15° C above the air temperature in shade and reached maximum values of about 32° C. At air temperatures (in sun) exceeding 29° C, toads maintained body temperatures below 32° C by evaporative cooling. Following heliothermic heating during the moring toads retreated to the shade, thereby decreasing body temperature below air temperature. Under overcast sky toads remained exposed during the whole day displaying body temperatures at or slightly above ambient levels. Quantitative models to predict the core temperature of toads under the different weather conditions demonstrated that the substrate temperature was the main energy source accounting for 64.6–77.9% of total variance whereas air temperature was of minor importance (1.5–4.4%). The unexplained variance was probably due to evaporative cooling. The volume of urine stored into the urinary bladder of toads varied diurnally; during basking in the morning hours most bladders contained large volumes of urine, whereas during the afternoon the bladders were mostly empty. The bladder contents probably serve as water reserves during basking when evaporative water loss was high. Toads preferred sites that provided shady hiding places as well as sun-exposed bare soil within a radius of 5 m. However, they frequently changed their centers of activity and moved to other sites in 20–70 m distance after periods of 2–5 days. The helio-and thigmothermic behaviour of the Andean toad permits the maintenance of high core temperature during morning which probably increases the digestion rate and accelerate growth. Evaporative cooling and preference of shady sites were employed to regulate body temperature below the morning levels in response to the constraints of water balance. Periodic changes between thigmothermic behaviour and locomotory activity during the night maintains body temperature above air temperature and prolongs the period of food uptake.Dedicated to Prof. Dr. H. Schneider on the occasion of his sixtieth birthday     

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 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.     

Water balance during saline imbibition in the Mongolian gerbil (Meriones unguiculatus)

1. There were few changes in the water balance of gerbils drinking 0.25 and 0.50 M NaCl solutions for 5 days. 2. Imbibition of 0.75 and 1.0 M saline resulted in some dehydration of the body fluids and considerable depletion of the neural lobe vasopressin store. 3. Although large amounts of NaCl were excreted, the maximum urine osmolality was considerably less than that found following water deprivation. 4. The imbibition of 1.0 M saline caused similar changes in water balance to water deprivation showing that little, if any, water was gained from this solution.     

Angiotensin II elicits water seeking behavior and the water absorption response in the toad Bufo bufo

Fully hydrated toads, Bufo bufo were acclimated to a simulated terrestrial habitat, with access to shelters and water. To get from the shelters to the water, the toads had to walk across the pan of an Ohaus balance and the body weights were recorded on a computer. Toads were placed inside shelters immediately following injection of human angiotensin II (A II), Thr(8)-saralasin, or Ringer's in the dorsal lymph sac, and their behavior was recorded continuously by video surveillance. The injection doses were 1-100 microg/100 g body weight A II and 100 microg/100 g body weight saralasin dissolved in 0.1 ml Ringer's; control animals received the same volume of Ringer's. The latency from injection to the initiation of water absorption behavior (WR) was significantly shorter in both A-II- and saralasin-injected toads, compared to controls. A-II- and saralasin-injected toads also spent significantly more time in the water than controls. The bladder depots when WR was terminated were significantly larger in A-II- or saralasin-injected toads than in controls. The stimulatory action of Thr(8)-saralasin, an antagonist of A II in mammals, on WR behavior in B. bufo suggests differences in receptor structure and/or receptor distribution between amphibians and mammals.     

Seasonal effects of dehydration on glucose mobilization in freeze-tolerant chorus frogs (Pseudacris triseriata) and freeze-intolerant toads (Bufo woodhousii and B. cognatus)

It has been hypothesized that freeze-tolerance in anurans evolved from a predisposition for dehydration tolerance. To test this hypothesis, we dehydrated summer/fall-collected and winter acclimated freeze-tolerant chorus frogs and dehydration-tolerant, but freeze-intolerant, Woodhouse's and Great Plains toads to 25% and 50% body water loss (BWL). Following treatments, we measured glucose, glycogen, and glycogen phosphorylase and glycogen synthetase (summer/fall only) activities in liver and leg muscle. Hepatic glucose levels were not significantly altered by dehydration in either summer/fall-collected frogs or toads. Conversely, winter acclimated frogs did show an increment (2.9-fold) in hepatic glucose with dehydration, accompanied by a reduction in hepatic glycogen levels. Winter acclimated toads did not mobilize hepatic glucose in response to dehydration. Further, hepatic glycogen and phosphorylase activities did not vary in any consistent manner with dehydration in winter toads. Mean leg muscle glucose values were elevated at 50% BWL relative to other treatments, significantly so compared to 25% BWL for summer/fall-collected frogs. The pattern of hepatic glucose mobilization with dehydration in winter frogs is consistent with that in other freeze-tolerant frog species, and provides additional support for the hypothesis that freezing tolerance evolved from a capacity for dehydration tolerance. However, the lack of hepatic glucose mobilization in response to dehydration in fall frogs suggests that a seasonal component to dehydration-induced regulation of glucose metabolism exists in chorus frogs. Furthermore, the absence of a dehydration-induced mobilization of hepatic glucose at both seasons in toads suggests that this dehydration response is not universal for terrestrial anurans.     

Water storage compromises walking endurance in an active forager: evidence of a trade-off between osmoregulation and locomotor performance

Trade-offs between locomotor performance and load-carrying in animals are well-established and often result from requisite life processes including reproduction and feeding. Osmoregulation, another necessary process, may involve storage of fluid in the urinary bladder of some species. The purpose of this study was to determine whether storage of urine in the urinary bladder reduces walking endurance in an actively foraging lizard. The results of our paired-design study indicate that the volume of fluid stored in the urinary bladder (36.5+/-1.6 ml) contributed a significant load (9.2% of body mass) to the lizards. This load resulted in a disproportionate 24.5+/-2.8% decrement in walking endurance. Specifically, Gila monsters walked at a fixed pace for a significantly shorter duration when the urinary bladder contained fluid (26+/-2.0 min) compared to when the bladder was empty (34.3+/-2.3 min). Since fluid stored in the bladder contributes to osmoregulation in this species, our results indicate the presence of a trade-off between osmoregulation and endurance in Gila monsters. Bearing other loads (e.g., a clutch or meal) influences the evolution of life-history traits and foraging strategy; thus the negative effect of fluid storage on endurance may also have evolutionary implications.     

Effects of angiotensin II and bladder condition on hydration behavior and water uptake in the toad,Bufo woodhousei

• 1.1. Water absorption response (WR) behavior and water weight gain were examined in hydrated toads, Bufo woodhousei, treated with angiotensin II (All) or with a control Ringer's solution. The effects of urinary bladder condition (ad lib. bladder urine or empty bladder) were examined concurrently.
• 2.2. Toads treated with All (100μg/100g body weight), spent more time in WR posture and absorbed more water than Ringer's-injected toads.
• 3.3. Toads with empty bladders maintained WR posture for longer periods of time and gained more weight than toads whose bladders were not emptied.
• 4.4. The effects of All and bladder urine on water absorption by B. woodhousei appear to be separate and additive.

     

Volume regulation in salt-acclimated toad (Bufo viridis): the role of urea and the urinary bladder

Body water (weight) was studied in the euryhaline toad Bufo viridis during high salt (500 mOsm NaCl) acclimation. Plasma osmolality was greatly increased upon salt acclimation mainly by urea, and was always hyperosmotic to the ambient solution. Water content was regulated quite efficiently in slowly acclimated undisturbed toads. Repeatedly catheterized toads behaved like osmometers when transferred to hyperosmotic solutions. Total urea loss was greatly reduced in salt acclimated toads, suggesting urine was not voided under these conditions. It is concluded that urea accumulation, inhibition of the urine voiding response and the urine in the bladder are the principal factors involved in volume regulation under conditions of salt acclimation.     

Angiotensin II alters blood flow distribution in amphibians.

In toads, angiotensin II (ANG II) induces the water absorption response (WR) during which the seat patch (pelvic+inner-thigh skin) is pressed to a wet substrate from which water flows osmotically into the animal. Since ANG II is a potent vasoconstrictor, it has the potential to redistribute blood flow. To determine the regional circulatory effects of ANG II, we used microsphere methods to measure relative changes in blood flow to several skin regions and other organs before and after ANG II administration in terrestrial toads and aquatic bullfrogs. In toads, after ANG II administration, seat patch and bladder blood flow increased by 264.2%+/-197.6% and 287.2%+/-86.7%, respectively (P<0.05), while dorsal and pectoral skin flow decreased by 48.0%+/-19.4% and 21.3%+/-25.4%, respectively (P<0.05). In bullfrogs, ANG II caused no significant changes in blood flow. Our results support our hypothesis that, in toads, ANG II increases and decreases blood flow to regions of the body associated with water gain and water loss, respectively.     

Aquatic and terrestrial locomotory energetics in a toad and a turtle: a search for generalisations among ectotherms

Murray short-necked turtles were trained to walk on a motorised treadmill and to swim in a recirculating flume. Through filmed records, the frequency of limb movement and the time that thrust was directed against the substrate were measured. The animals wore masks when walking and accessed air when swimming from a ventilated capsule placed on top of the water surface. Measurement of the exhalant O(2) and CO(2) levels from these devices enabled the measurement of metabolic rates. Equivalent data were obtained from swimming and hopping cane toads, although repeatable measures of limb frequency and contact times were not obtained due to the intermittent form of locomotion in this species. Comparing the cost of transport, the energy required to transport a mass of animal over a unit distance, with other animals showed that toads do not have a cheap form of terrestrial locomotion, but turtles do; turtles use half the cost predicted from their body mass. This economy of locomotion is consistent with what is known about turtle muscle, the mechanics of their gait, and the extremely long contact time for a limb with the substrate. Swimming in toads is energetically expensive, whereas turtles, on the basis of mass, use about the same energy to transport a unit mass as an equivalent-size fish. The data were compared with the predictions of the Kram-Taylor hypothesis for locomotory scaling, and walking turtles were found to provide a numerical fit. The data show that both terrestrial and aquatic locomotory energetics in toads are generally higher than predictions on the basis of mass, whereas in turtles they are lower.     

Elevated plasma osmotic concentration stimulates water absorption response in a toad.

The water-seeking behavior (WR) of toads (Bufo viridis) was investigated. Fully hydrated toads that are allowed free choice of wet or dry filter paper voluntarily and spontaneously select to sit on water-soaked paper at a regular frequency during trials. Dehydration of bladder-emptied toads by 14% elicits WR in all animals. Injection of aldosterone or angiotensin-I reduced the dehydration threshold to 7% weight loss. WR frequency increased when plasma osmolality was elevated by injection of NaCl or other solutes (both ionic and non-ionic). Only urea, to which cell membranes are highly permeable, was the exception that did not produce this response. The increase in WR frequency induced by elevated plasma osmolality was augmented by injection of aldosterone or angiotensin-I. In vivo water uptake, measured in a water bath, was increased by an NaCl or oxytocin injection, but not by aldosterone. It is concluded that elevated plasma osmolality induces an increase in WR frequency that is separate and prior to the water uptake process. Different hormones are involved in each step.     

Kidney and bladder function in a uricotelic treefrog (Phyllomedusa sauvagei)

Summary Phyllomedusa sauvagei, a xeric adapted treefrog, excretes large amounts of nitrogen as urate when fed insects, even when deprived of additional water. Most terrestrial anurans produce urea which they do not excrete when they are deprived of water. We investigated the differences in renal function underlying the unusual excretory capacities ofP. sauvagei. Glomerular filtration rates (GFR) were measured inP. sauvagei in water and when deprived of water, except that in food, for up to 27 days. For comparison a toad (Bufo boreas) was studied in water and during water deprivation. In water both species produced 30–40 ml urine kg^–1 h^–1 and resorbed only ca. 50% of the filtrate. With water deprivation, GFR rapidly approached zero inB. boreas, but remained high (20–40 ml kg^–1 h^–1) inP. sauvagei despite reductions in urine production of up to 100-fold. During water deprivation inP. sauvagei, urate excretion was between 250–300 moles kg^–1 h^–1 and 90% of this reflects net tubular secretion. Urate clearances were similar to those of para-amino hippurate, indicating effective removal of urate from the peritubular circulation. Urea, sodium and chloride showed net fractional resorptions of 98–99%, and 85% of the potassium was resorbed. At low rates of urine production, urine to plasma (U/P) ratios for inulin in bladder urine were 20–100 whereas those for ureteral urine were ca. 10. The urinary bladder also functions as a water reserve during dehydration.     

笼养白头叶猴夏季水分摄入与消耗的初步研究

叶片中的水分含量是白头叶猴水分的主要来源,占水分需求总量的８３．６８％,其它的１６．３２％来自于自由水。在笼养条件下,白头叶猴可饮用自来水,野生状态下,动物饮用露水或雨水。尽管饮水行为不是每天都有,但能经常观察到,特别是在夏季雨后．除了体表和呼吸系统的水分蒸发外,白头叶猴通过尿和粪便丢失的水分含量分别是４４．６８％和５５．３２％。     

Effect of salt depletion on sodium concentration in serum and urine of Bufo chilensis. Evidences for increased levels of neurohypophysial principles in their plasma

Salt-depleted toads Bufo chilensis were compared with animals maintained in NaCl solution and a control group with respect to Na+ content in serum and urine. Plasma hydro-osmotic activity of the animals was measured by increased water transfer across the isolated urinary bladder of the frog (Caudiverbera caudiverbera). Sodium in serum is not affected by pre-adaptation in distilled water. Urine Na+ is markedly reduced. Plasma from depleted animals increases water transfer across the isolated urinary bladder. Immersion in NaCl solution did not have this effect. An increase in neurohypophysial hormones in the blood of the animals is postulated.     

Deltamethrin-induced deregulation of the water balance in the migratory locust,Locusta migratoria

1. Several insecticides were tested for their ability to induce a water deregulation in the larval migratory locust. All of them provoked an accelerated dehydration (when compared to sham-operated insects). Deltamethrin and baygon were the most potent.2. This enhanced dehydration due to deltamethrin in adult locust resulted from an increase in the water loss through the feces. This increase was not due to a direct effect of deltamethrin on urine production by the Malpighian tubules but to a hormonal deregulation.3. Intoxicated insects produced large amounts of the vasopressin-like insect diuretic hormone. This higher synthesis activity occurs within the hours following the insecticide injection and is accompanied by an increase in water loss.4. These hormonal and metabolic modifications are transient. Hormonal level and diuresis rate both return to the basal levels 7 hr after the insecticide injection.