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.     

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.     

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.     

Perch color preference in juvenile green tree pythons,Chondropython viridis

Green tree pythons, Chondropython viridis, are polymorphic for color as juveniles, commonly being primarily yellow or brown until becoming mostly green at about 1 year of age. We tested the hypothesis that the different morphs arose as a result of selection for differential background matching, yellow morphs selecting light-colored backgrounds, and brown morphs selecting dark-colored backgrounds. Twelve yellow and eight brown morphs were placed repeatedly in individual testing enclosures and allowed to choose between black and white or yellow and brown halves of a t-perch. Trials showed that both color morphs preferred dark over light perches. We tentatively suggest that individuals chose dark-colored perches for purposes of concealment. © 1994 Wiley-Liss, Inc.     

Plasma osmolality, urine composition and tissue water content of the toad Bufo viridis Laur. in nature and under controlled laboratory conditions

The compositions of plasma and urine were studied in toads (Bufo viridis) which were collected from three locations in Israel, and compared with toads which were kept under constant laboratory conditions for nearly 2 years. Plasma osmolality was rather constant (over 310 mOsm kg-1 H2O) during the whole year in the active toads. Urea was the most variable osmolyte in the plasma, and accounted for the higher osmolality in southern population. Urine osmolality fluctuated in a circannual fashion both in freshly captured and in the toads under constant laboratory conditions. Water content of the tissues was constant throughout the year, independent of the plasma osmolality. It is concluded that high plasma urea concentration and the excretory system (kidneys and the urinary bladder) are important in sustaining constant plasma osmolality in active toads. Both mechanisms change annually and form the basis for the high terrestriality of this species.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The vitelline envelope-to-fertilization envelope transformation in the toad Bufo arenarum.

An investigation of some changes associated with the transformation of the vitelline envelope into the fertilization envelope in the egg of the toad Bufo arenarum is reported here. In most of the experiments described, the parameter used to demonstrate these changes was the stability of structural integrity of isolated envelopes when submitted to different agents and conditions. The envelopes used for this purpose exhibited a high degree of purity and remained apparently unaltered by the isolation procedure. As a quantitative method to ascertain their solubility rate, the release of uv-absorbing materials into solution was determined. Compared to the vitelline envelope, the fertilization envelope has proven to be less soluble in water, more stable in the presence of the chaotropic ion thiocyanate, and less susceptible to digestion in the presence of sperm lysin, trypsin, and pronase. In Bufo arenarum, as in other species, the vitelline envelope appears to be composed of glycoproteins. In contrast to previous results, however, disulfide bonds do not seem to be involved in their structural integrity. Thus, experiments carried out with isolated envelopes as well as with envelopes in situ have demonstrated a lack of effect of disulfide bond breaking agents on envelope stability. Evidence is presented suggesting that the solubility of envelopes in mercaptan solutions, as reported by other laboratories, is likely to be the expression of artifactual results.     

Central thermoregulatory effects of lactate in the toad Bufo paracnemis.

Hypoxia induces a regulated decrease in body temperature (Tb; anapyrexia) in organisms ranging from protozoans to mammals, but very little is known about the mechanisms involved. Several candidates have been suggested to mediate hypoxia-induced anapyrexia, among them lactate, which is a classical compansion of hypoxic stress in vertebrates. The present study was designed to assess the central thermoregulatory effects of lactate in Bujo paracnemis. Toads equipped with a temperature probe were tested over a thermal gradient (10-40 degrees C). Lactate injected systemically (4.0 mmol kg-1) caused a significant reduction of Tb from 24.6 +/- 2.1 to 17.4 +/- 3.9 degrees C. To assess the role of central thermoregulatory mechanisms, a lower dose (0.4 mmol kg-1) of lactate was injected into the fourth cerebral ventricle or systemically. Intracerebroventricular injection of lactate caused a similar decrease in Tb, whereas systemic injection caused no change. The data indicate that lactate may play a role in hypoxia-induced anapyrexia in central rather than peripheral sites.