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.     

Effect of the venom of the toad Bufo viridis Laur. on cardiac hemodynamics in dogs

The cardiotonic effect of the secretion of the toads Bufo viridis Laur. skin glands and discharged bufadienolidines has been studied in the experiments on dogs under anaesthesia. It has been shown that the toad poison and bufadienolidines administered intravenously produce a marked cardiotonic and vasotonic effect, increasing the intraventricular and aortal pressure, the rate of pressure growth in the ventricles and the contraction index of the myocardium. The cardiotonic effect of poisons has also been noted in the model study of cardiac insufficiency caused by the bandaging of the coronary artery, it has greater effect than that of the cardiac glycoside corglycon.     

A shift in prey color preference in the green toad Bufo viridis Laur. after food satiation]

In the behavioural experiments when showing simultaneously coloured stimuli (food targets) and also in choosing between two stimuli, red and blue, it had been shown with a high level of reliability that the hungry Bufo viridis which had not been fed for at least a week, in the overwhelming majority of cases chose the red-coloured food targets. Within 1-3 days after an abundant feeding, in the same animals the changes occurred in their preferences of the prey colour. The percentage of choosing the blue targets by the satiated toads significantly increases. In a separate series of experiments had been demonstrated that the changing motivation is conditioned by colour, since hungry and satiated Bufo viridis under conditions of simultaneous demonstration of four stimuli, black and three different grey stimuli, chose exclusively the black stimulus.     

Tissue osmolytes and body fluid compartments in the toad Bufo viridis under simulated terrestrial conditions

Summary Toads (Bufo viridis) were kept on soil without access to free water (simulated terrestrial conditions) for over 12 weeks. Body water compartments were estimated using the dilution method (inulin and Evans Blue). They were found to remain fairly constant after a period of adjustment which lasted 1–2 weeks. In particular, plasma volume was closely controlled. Plasma osmolarity increased to over 1000 mOsm · 1^–1 accompanying a large increase in its urea concentration. NaCl also increased, while potassium remained constant. Tissue (liver and skeletal muscle) water content did not change much and electrolytes were kept constant. Tissue water urea concentration seemed to equilibrate with that of the plasma. Urine osmolarity, which was hypotonic during water access, became nearly isosmotic and correlated with the plasma following transfer onto soil. Urine urea concentration correlated with the plasma in the terrestrial conditions, potassium was greatly elevated, sodium increased to some extent, and chloride hardly changed. The efficient osmoregulatory mechanisms for the control of distribution of body water sustain normal physiological functions.     

Urea production and accumulation in the green toad, Bufo viridis

The toad, Bufo viridis , can live for several months without access to free water, absorbing soil-bound water down a water-potential gradient created, mainly, by accumulating urea in its body fluids. We investigated if the retention of urine was sufficient to account for the rate of accumulation or if an increased rate of urea production was needed in order to do so. The basal rate of urea production in unfed animals in the absence of osmotic stress was estimated by two methods; first, analysis of the bathing medium and, secondly, collection and analysis of urine at two-hourly intervals. This was then repeated with animals fed a weight-maintaining diet. Generally similar results were obtained by either method in both fed and unfed animals, although higher urea production rates were found in the former. Although it had been planned to apply the short interval method to toads with free access to water, the control condition for toads transferred to soil, it proved to be impracticable. Some animals did not bathe for almost a day, during which time minute quantities of urine were obtained. Larger volumes were only produced during or after bathing. Consequently, animals which were partially immersed in water were substituted as controls. Total urea content was determined in these and in toads after a week on soil. The calculated increase was compared to that which could be expected from urine retention. It was found that urea accumulated at more than twice the predicted rate. When rates of accumulation were calculated over longer periods, urine retention alone was sufficient to account for them within three weeks on soil, the usual period required for acclimation. We concluded that B. viridis increased its rate of urea production only for a short period, until a favourable water potential gradient was achieved.     

Effects of the ecological conditions on morphological variations of the green toad, Bufo viridis, in Turkey

Ten populations of the green toad, Bufo viridis, from three areas having different climatic regimes were compared. Multivariate analyses of morphological characteristics revealed three distinct populations of green toads: the first group inhabiting the lowland and humid localities was characterized by a small body size, the second group occurred at higher altitudes and in desert areas had a significantly larger body size, while the third group from the Mediterranean localities was characterized by a large tympanum, a longer tarsus and short first toes. Interestingly, the specimens from Giresun Island were clustered with the second group with a similar pattern of body size. Differences in the morphological characteristics among populations were positively associated with ecological conditions, and not with geographic distance. Some characteristics previously used for taxonomic purposes in B. viridis were shown to be associated with local climatic conditions.     

Digitalis-like compounds in the toad Bufo viridis: tissue and plasma levels and significance in osmotic stress.

Digitalis-like compounds (DLC), constituents of animal tissues, are possible regulators of the Na+, K(+)-ATPase implicated in water and salt homeostasis. The distribution of DLC in the toad (Bufo viridis) was determined following methanol extraction and partial purification. DLC highest levels were found in the skin but it was also detected in the plasma and many internal organs. Short term (hours) exposure of the toad to hypertonic shock (1.5% NaCl) induced an increase in plasma osmolarity due to an increase in Na+ and Cl- levels. This treatment induced a transient, three fold, increase of DLC levels in the brain and transient reduction of its levels in the ventral skin. Acclimation of the toads to burrowing conditions for six weeks resulted in an increase in plasma osmolarity due to a large increase in plasma urea with a small increase in ion concentrations. Under these conditions DLC levels in the dorsal skin increased by 100% without alteration of its levels in the plasma, brain and ventral skin. DLC levels in the toad brain of control animals, showed a significant dependence on season, being highest in the summer and lowest in the winter. DLC levels in the skin peaked in May while the levels in the plasma were season independent. The changes in DLC levels induced by the short- as well as long-term perturbations in the animal environmental salinity together with the seasonal differences suggest that DLC in the toad is involved in water and salt homeostasis of these animals, but may also participate in other unknown functions.     

Effect of bromocriptine on the larval skin of the green toad, Bufo viridis viridis leurenti

In the premetamorphic larval green toad, B. viridis viridis, as in other anurans, the skin is made up of a fibrous dermis and an epidermis of stratified epithelium. The effects of bromocriptine, an antiprolactin drug, on the premetamorphic skin of B. viridis viridis was examined. Bromocriptine, dissolved in rearing water at four different concentrations, induced a number of changes in the skin of treated tadpoles. In rough sequence of appearance, these changes include: retraction ofthe melanocyte dendrites, synchronous burst ofthe apical vesicles of the superficial epithelial cells, gradual disappearance of the melanosomes from the epithelial cells and widening of the intercellular spaces. In addition, macrophages appeared in the superficial dermis amongst the retracted melanocytes. White crystals were observed on the skin surface and similar crystals were ingested by the macrophages. Prolonged treatment with bromocriptine resulted in hypertrophy and extraction of some epidermal cells. Deep melanocytes of the mesenteries were not affected by bromocriptine-treatment indicating that the drug did not penetrate deep into the tadpole tissue. Whether the macrophages observed in the dermis were recruited from deeper tissues or were converted melanocytes is another issue in need of study.     

NaCl adaptation in Rana ridibunda and a comparison with the euryhaline toad Bufo viridis.

The physiological adaptation of the frog Rana ridibunda to saline environment was studied. It was found that blood was always hypertonic to the external solution, but at the highest salinity tolerated (i.e. 300 mOsM) the osmotic gradient across the skin was nearly abolished. Water uptake by the living frog remained unchanged, whereas sodium transport across the skin decreased markedly. Neurohypophyseal hormone increased water uptake and sodium transport to levels similar to those in tap water frogs. Water content of the tissues was not affected by saline adaptation, although it varied appreciably under acute conditions. Oxygen consumption increased in dehydrated frogs, but not in adapted ones. The results are discussed and compared to the euryhaline toad Bufo viridis; the importance of high urea levels for high salt adaptation is stressed.     

The ecological significance of burrowing behaviour in the toad (Bufo viridis)

Summary The burrowing behaviour of the toad Bufo viridis was studied in the laboratory on soil containing 11–13% moisture. These toads usually excavate shallow, temporary burrows from which they emerge intermittently. Time spent subsurface increases with rising ambient temperature in the range of 18–30° C. In these conditions, water loss is effectively reduced and a new water balance is achieved. On return to water access, after two months on soil, the toads rapidly regain only that amount of water lost previously. It is concluded that this temperature-sensitive burrowing behaviour is an important factor in these animals' successful survival in semi-arid conditions.     

Binding sites of opiates and endogenous opioids in the oocytes of the toad Bufo viridis

The binding of labelled naloxone, morphine and (D-Ala2,D-Leu5)enkephalin (DADL) to oocyte membranes of the toad Bufo viridis was investigated. The opiate antagonist naloxone binds to the membranes much more effectively than morphine or DADL. The binding of [3H]naloxone is reversible and saturating. The bound [3H]naloxone is readily replaced by unlabelled naloxone or bremazocine (kappa-agonist), far less effectively by morphine (mu-agonist) and SKF 10.047 (sigma-agonist) and is not practically replaced by DADL (delta-agonist), beta-endorphin (epsilon-agonist) and other neuropeptides. Analysis of experimental results in Scatchard plots revealed two types of binding sites with a high (Kd = 15 nM) and low (Kd = 10(3) nM) affinity for naloxone. The number of sites responsible for the binding of naloxone possessing a high affinity is 16 pmol-/mg of oocyte homogenate protein, i.e., 20-50 times as great as in the toad or rat brain. Trypsin and p-chloromercurybenzoate decrease the binding of [3H]naloxone. The oocyte extract is capable of replacing the membrane-bound [3H]naloxone, on the one hand, and of inhibiting the smooth muscle contracture of the rabbit vas deferens, on the other. This inhibition is reversed by naloxone and can also be induced by bremazocine, but not by morphine, DADL and SKF 10.047. In all probability oocytes contain compounds that are similar to opiate kappa-agonists. It may also be possible that these compounds mediate their effects via specific receptors and are involved in the control over maturation of oocytes and early development of toad eggs.     

Osmoregulation of the cane toad, Bufo marinus, in salt water

Adult cane toads, B. marinus, survived in salinities up to 40% sea-water (SW). Pre-exposure to 30, then 40% SW, increased the survival time of toads in 50% SW. Plasma from toads acclimated to salt water is hyperosmotic to the environment--a result of increased plasma sodium, chloride and urea concentrations. When toads were placed in tap-water and 20% SW, all significant changes to plasma sodium, chloride, urea and osmotic pressure occurred within the first 2 days of exposure. When toads were placed in 30 and 40% SW environments, the increases in plasma sodium and chloride concentrations occurred within the first 2 days of exposure while urea and total osmotic pressure continued to rise until some time between 2 and 7 days exposure.     

Renal response of euryhaline toad (Bufo viridis) to acute immersion in tap water, NaCl, or urea solutions

Green toads (Bufo viridis) were acclimated to either tap water, 230 mOsmol NaCl kg-1 H2O (saline), 500 mOsmol NaCl kg-1 H2O (high saline), or 500 mmol L-1 urea. Renal functions for each acclimation group were studied on conscious animals that had one ureter chronically catheterized. Reciprocal immersion of tap-water- and saline-acclimated toads in the opposite solution did not stress the animals osmotically, and plasma osmolality increased or decreased by no more than 15%. However, urine osmolality and ionic composition changed immediately and profoundly on exposure to the other solution. Exposure of tap-water-acclimated toads to saline decreased urine flow by 30%, whereas the reciprocal immersion led to an increase of 30%. Immersion of tap-water-acclimated toads in high saline led to immediate cessation of urine flow, whereas immersion of 500 NaCl- or urea-acclimated toads in tap water led to a large increase in urine flow, with an overshoot that lasted 10 h (as a result of either salt or urea diuresis). Urine flow then stabilized at a level 5-6 times higher than the value attained at high-salt environment. On immersion of 500 urea-acclimated toads in 500 NaCl, urine flow doubled, accompanied by a change in ion composition, without change in the osmolality. In all experimental conditions, plasma potassium concentration was maintained within a narrow range. The results show that the toad's kidneys contributed efficiently both to osmo- and ionoregulation in a wide range of ambient solutions.     

Taurine in toad brain and blood under different conditions of osmolality: An immunohistochemical study

The concentrations of taurine in blood and brain regions of the toadBufo boreas have been measured. Most of these values are considerably lower than those found in mammals. Using an antibody prepared against conjugated taurine, the distribution of taurine in three brain regions of the toad has been visualized. The possible osmoregulatory functions of taurine have been investigated by making toads hyper- or hypo-osmotic in vivo. Induction of hypoosmolality is accompanied by a massive taurine tide in blood plasma, but has no immediate effects upon the taurine concentrations in the brain areas studied. However, histochemical visualization indicates a marked redistribution of taurine between cellular components and extracellular space of brain tissues. This may indicate that taurine has an osmoregulatory function in brain tissue under hypo-osmotic conditions. Hyperosmolality results in no elevation of the taurine concentration in blood plasma of toads, but rather in a very gradual decline of total plasma taurine content over a prolonged time period. Histochemical studies reveal little change in frontal cortex after 1 hour but deeper staining of many neurons in optic lobe accompanied by greater staining in the extracellular fluid. By 3 hours there is a depletion of taurine from all compartments of cerebral cortex tissues. No evidence of any prolonged direct osmoregulatory role for taurine is indicated under hyperosmotic conditions. A possible indirect osmoregulatory function of taurine is discussed.Special issue dedicated to Dr. Claude Baxter.     

Histochemical investigations on the secretory cells in the oesophagogastric tract of the Eurasian green toad,Bufo viridis

The secretory cells of the oesophagogastric tract of the Eurasian toad, Bufo viridis, were examined using standard histochemical methods and lectin histochemistry. Two goblet cell types were found in the oesophageal epithelium, differing in their morphology and the histochemical features of the secretory granules. These contained mainly acidic glycoconjugates, both sulphated and carboxylated, and a small amount of pepsinogen. Type I goblet cells contained stable class-III mucosubstances, which were absent in Type II. No pluricellular oesophageal glands were found. The oesophagogastric junction had a superficial epithelium similar to that of the oesophageal epithelium, with alveolar pluricellular glands, secreting stable class-III mucins, and few oxynticopeptic cells. The gastric mucosa presented secretory cells both in the surface epithelium and in the gastric glands. Superficial and foveolar cells produced neutral mucins with Gal1,3GalNAc residues. Neck cells, oxynticopeptic cells and endocrine cells were found in the gastric glands. Neck cells produced stable class-III mucosubstances. A functional gradient was observed in the oxynticopeptic cells from the oral to the aboral fundus, with a decrease in pepsinogen secretion towards the aboral fundus and a possible increase in HCl secretion. In the pyloric mucosa, the oxynticopeptic cells disappeared and the glands produced only neutral mucins, without stable class-III mucosubstances.     

The effect of salt acclimation of the water uptake and osmotic permeability of the skin of the toad (Bufo viridis, L.)

1. Water uptake in vivo, and water fluxes across the isolated skin were studied in salt (NaCl) acclimated toads. 2. Water uptake of acclimated toads maintained in the solution of acclimation, decreased with the environmental salinity. 3. The osmotic water permeability (Pos) of the skin increased upon salt (NaCl) acclimation, both in vivo and in vitro. 4. Pos of the skin of toads acclimated to non-permeant solutes such as sucrose (230 mmol/l) or mannitol (400 nmol/l), was greatly reduced. 5. Oxytocin (syntocinon) increased the Pos both in tap water and salt acclimated toads. In high salt (greater than 200 mmol/l NaCl) acclimated toads however, the increased Pos and water flux at larger osmotic gradients, could not be stimulated further by the hormone. 6. The adaptive nature of the selective changes in the permeability properties of the skin under salt acclimation conditions is discussed.