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.     

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.     

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.     

The primary immune response of the green toad (Bufo viridis) to challenge with Crithidia fasciculata

The primary immune response of the green toad (Bufo viridis) following immunization with Crithidia fasciculata choanomastigotes was studied. Lysins, agglutinins, and antibodies detectable by enzyme-linked immunosorbent assay (ELISA) were first detected in the sera of immunized animals one week after injection. The antibody titers increased to significant levels (P less than 0.01) and maximum values were reached seven weeks post-immunization. The stimulated immunoglobulins were antigen-specific, partially heat-labile, sensitive to the reducing agent dithiothreitol, possessed precipitin activity, effectively fixed complement and exhibited an electrophoretic mobility similar to the gamma-globulins of human serum. On this basis, it is probable that the antibody produced during the primary response in green toads is high molecular weight IgM. Increases in serum lysozyme levels paralleled the rise of antibody titers. Overall, the lysozyme concentration increased two-fold compared to the appropriate controls. This is the first report of the immune response in amphibians to experimental injection with protozoan parasites and the use of the ELISA technique to detect antibodies in amphibian sera.     

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.     

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.     

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.     

Changes in ionic conductances and in sensitivity to amiloride during the natural moulting cycle of toad skin (Bufo viridis,L.)

Summary The resistance of the apical membranes of toad skin (Bufo viridis) was measured during its natural moulting cycle using a fast flow technique. The skin behaved in all periods of the moulting cycle as a nearly perfect sodium electrode. In the presence of amiloride (10^–4 M), the total resistance of the same skin was identical with solutions which contained either sodium or potassium. The resistance of the skin with potassium was sensitive to amiloride in the period just after moulting. The resistance of skins which were made shunted by treating them with urea on the outside was insensitive to amiloride in solutions containing potassium; a small effect was still observed with sodium. It is suggested that the transient sensitivity to amiloride, with potassium, is the result of differentiation of the sodium specific sites at the apical membranes of the skin.     

New polymorphic microsatellite markers and development of mitotyping primers for West Mediterranean green toad species (Bufo viridis subgroup)

We report new polymorphic microsatellites for three species of Palearctic green toads (Bufo viridis subgroup): 10 in B. balearicus and seven each in B. siculus and B. boulengeri. Diversity at these loci, measured for 27 B. balearicus, 23 B. siculus and 11 B. boulengeri, ranged from low to high (two to 10 alleles). Mitotyping primers, specific to the control region, which allow fast screening of parapatric Sicilian endemic B. siculus and Italian mainland-origin B. balearicus, were developed.     

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.     

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.     

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.     

Effect of alpha-2 adrenergic receptor stimulation on short-circuit current across isolated skin of the toad Bufo arenarum

1. The effect of the alpha-2 adrenergic agonist clonidine on short-circuit current (SCC) across isolated skins of Bufo arenarum toads was investigated. 2. Clonidine inhibited basal SCC in a dose-dependent manner. 3. Blockade of the effect of clonidine on basal SCC by the selective alpha-2 antagonist yohimbine supports the hypothesis that the inhibitory effect is mediated by the stimulation of alpha-2 adrenergic receptors. 4. The fact that the inhibitory effect of clonidine is higher in skins with spontaneous positive SCC than in the negative ones, and that the alpha-2 agonist was unable to alter amiloride-induced negative SCC suggests that the inhibitory effect of clonidine may probably be mediated by inhibition of sodium transport.