Experimental exposure of Canadian toads to Basidiobolus ranarum

Experimental transmission of the fungus Basidiobolus ranarum was induced in two treatment groups of Canadian toads (Bufo hemiophrys) and caused a fatal mycotic dermatitis. Seven of 10 (70%) toads that had their ventral skin mildly abraded and exposed to B. ranarum developed hyperemia, and sloughing of their ventral skin and died. Toads with abraded ventral skin or exposure to infected skin also were affected statistically at a higher rate than those with abraded skin and exposure to pure cultures of B. ranarum inoculated into their water source. Of toads showing clinical disease, B. ranarum was identified by both impression smears and histology in all cases, but not from toads that appeared clinically healthy. The organism was cultured from 5 of 7 (71%) toads with clinical disease but not from any toad that appeared clinically healthy (n = 28). This study documents methods of experimental transmission of B. ranarum, an organism responsible for causing a mycotic dermatitis that is fatal to toads.     

Mucormycotic dermatitis in captive adult Wyoming toads

During late May 1995, 50 adult captive endangered Wyoming toads (Bufo baxteri) were brought out of hibernation. Approximately 3 to 10 days after hibernation emergence, all toads were hormonally induced to breed, and paired. Each pair was placed in their own breeding tank. Four toads developed clinical signs of disease which included lethargy and multiple (4 to 12) small (2 mm) raised hyperemic nodules with white fuzzy caps on the ventral skin. The condition progressively worsened until death occurred, within 3 to 6 days. Mycotic dermatitis caused by Mucor sp. was diagnosed in the four toads through histology and isolation of the organism. This is the first case report of a Mucor sp. causing a fatal dermatitis in an amphibian without significant inflammatory response and without systemic involvement.     

Causes of mortality of the Wyoming toad

Wyoming toads (Bufo baxteri) that died from January 1989 to June 1996 were submitted to the Wyoming State Veterinary Laboratory (Laramie, Wyoming, USA) for postmortem evaluation. These consisted of 108 free-ranging toads and 170 animals from six captive populations. Ninety-seven (90%) of 108 free-ranging toad carcasses were submitted during September and October. From 1989 to 1992, 27 (77%) of 35 mortalities in the captive populations occurred in October, November, and December. From 1993 to 1996, mortality in captive toads occurred without a seasonal pattern and coincided with changes in hibernation protocols that no longer mimicked natural cycles. Cause of mortality was determined in 147 (53%) of the 278 cases. Mycotic dermatitis with secondary bacterial septicemia was the most frequent diagnosis in 104 (71%) of 147 toads. Basidiobolus ranarum was found by microscopic examination of skin sections in 100 (96%) of 104 of these mortalities. This fungus was isolated from 30 (56%) of 54 free-ranging and 24 (48%) of 50 captive toads. This research documents the causes of mortality for both free-ranging and captive endangered Wyoming toads over a 7 yr period.     

Dermatitis in captive Wyoming toads (Bufo baxteri) associated with Fusarium spp

From May 2007 to June 2008, 30 of 49 Wyoming toads (Bufo baxteri) kept at Omaha's Henry Doorly Zoo (Nebraska, USA) died showing clinical signs of ventral erythema, inappetance, lethargy, and delayed righting reflex. Treatment with antifungals and antibiotics was unsuccessful in all cases. Histopathologic analyses revealed dermatitis as the primary problem in 20 of 21 toads in which skin was examined. Fungal dermatitis was present in 17 toads, with hyphae approximately 1-3 μm in diameter, and parallel cell walls and frequent septations. In 14 animals, the fungal dermatitis was the main pathologic lesion. Several species of bacteria were associated with all cases. A few animals tested positive for Ranavirus using polymerase chain reaction. Fusarium sp. was consistently cultured from skin, feces, kidneys, and from powdered food provided to crickets. Four isolates were identified as Fusarium proliferatum, Fusarium oxysporum, Fusarium solani, and Fusarium verticillioides, which suggested a secondary role of fungi. A specific underlying cause of disease could not be found, although the roles of humidity and Ranavirus infection are discussed, along with the well-known susceptibility of bufonids to fungal dermatitis.     

Water potential receptors in the skin regulate blood perfusion in the ventral pelvic patch of toads

Blood cell flux (BCF) in ventral pelvic skin capillaries was measured in conscious unrestrained Bufo bufo, using a laser Doppler flowcytometer. Hydrated toads responded to water contact with a small but significant increase in BCF. Dehydration alone did not change the BCF in seat patch skin before water contact. However, water contact by dehydrated toads elicited a rapid 600% increase in BCF. The BCF and water uptake of dehydrated toads rehydrating in water declined over 2 h but remained significantly above the low, constant values measured in hydrated toads. Arginine vasotocin injection in hydrated toads did not change skin BCF, but water uptake increased, and urine production decreased. Injection of the beta -adrenergic agonist isoproterenol increased BCF in hydrated toads by 900% and also increased the rate of water uptake. These increases corresponded in magnitude and duration to the response to water contact observed in dehydrated toads. Injection of dehydrated toads with the beta -adrenergic antagonist propranolol significantly reduced both BCF and water uptake. These results are consistent with an autonomic reflex mediated by skin water potential receptors that regulate blood perfusion of ventral pelvic skin.     

Trypanosomes of Bufo americanus from northern Michigan

Two hundred one American toads (Bufo americanus) from northern Michigan were examined for blood trypanosomes. Three species, Trypanosoma bufophlebotomi, T. schmidti-like sp. and T. pseudopodia, had prevalences of 27, 16 and 1%, respectively. Cross experimental inoculations showed that T. bufophlebotomi from toads is not the same as T. ranarum found in frogs of the family Ranidae of this region.     

Cutaneous blood flow and water absorption by dehydrated toads

Blood cell flux (BCF) in ventral pelvic skin capillaries was measured in toads, Bufo woodhouseii and Bufo punctatus, using a chamber that allowed hydration behavior and water absorption to be observed concurrently in unrestrained animals. Dehydrated B. woodhouseii and B. punctatus placed on a rehydration solution significantly increased BCF relative to that on a dry surface in less than 2 min. Skin contact with a rehydration solution rather than dehydration alone is the primary stimulus for increased seat patch blood flow. In B. woodhouseii, the water absorption response was initiated after the increase in BCF had started but before maximum BCF was reached. BCF and water uptake across the ventral skin of both species placed on deionized water were not different from those of toads placed on 50 mM NaCl. Similarly, no significant correlation between BCF and rate of water uptake could be observed in dehydrated toads of either species. Angiotensin II (AII) injection in hydrated B. punctatus had no effect on BCF, suggesting that factors other than AII are responsible for the increase in blood flow upon water contact in dehydrated toads.     

Localization of water channels in the skin of two species of desert toads, Anaxyrus (Bufo) punctatus and Incilius (Bufo) alvarius

Anuran amphibians obtain water by osmosis across their ventral skin. A specialized region in the pelvic skin of semiterrestrial species, termed the seat patch, contains aquaporins (AQPs) that become inserted into the apical plasma membrane of the epidermis following stimulation by arginine vasotocin (AVT) to facilitate rehydration. Two AVT-stimulated AQPs, AQP-h2 and AQP-h3, have been identified in the epidermis of seat patch skin of the Japanese tree frog, Hyla japonica, and show a high degree of homology with those of bufonid species. We used antibodies raised against AQP-h2 and AQP-h3 to characterize the expression of homologous AQPs in the skin of two species of toads that inhabit arid desert regions of southwestern North America. Western blot analysis of proteins gave positive results for AQP-h2-like proteins in the pelvic skin and also the urinary bladder of Anaxyrus (Bufo) punctatus while AQP-h3-like proteins were found in extracts from the pelvic skin and the more anterior ventral skin, but not the urinary bladder. Immunohistochemical observations showed both AQP-h2- and AQP-h3-like proteins were present in the apical membrane of skin from the pelvic skin of hydrated and dehydrated A. punctatus. Further stimulation by AVT or isoproterenol treatment of living toads was not evident. In contrast, skin from hydrated Incilius (Bufo) alvarius showed very weak labeling of AQP-h2- and AQP-h3-like proteins and labeling turned intense following stimulation by AVT. These results are similar to those of tree frogs and toads that occupy mesic habitats and suggest this pattern of AQP expression is the result of phylogenetic factors shared by hylid and bufonid anurans.     

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.     

Blood parasites of amphibians from Algonquin Park, Ontario

During a 5 wk period beginning May 25, 1983, 329 amphibians, which included specimens of Rana catesbeiana Shaw, Rana clamitans Latreille, Rana septentrionalis Baird, Rana sylvatica LeConte, Hyla crucifer Wied, Bufo americanus Holbrook, and Plethodon cinereus Green, from Lake Sasajewun, Algonquin Park, Ontario, Canada were examined for blood parasites. The prevalences of species of Trypanosoma, Haemogregarina, Lankesterella, Babesiasoma, and Thrombocytozoons in these amphibians were determined. Two species of microfilaria (probably Foleyella spp.) and two intraerythrocytic forms, inclusions of an icosahedral cytoplasmic DNA virus (ICDV) and groups of rickettsial organisms, were also observed. The following are new host records: Trypanosoma ranarum (Lankester, 1871) in B. americanus; Trypanosoma ranarum (Lankester, 1871) in R. sylvatica; Trypanosoma pipientis Diamond, 1950, Babesiasoma stableri Schmittner and McGhee, 1961 and Thrombocytozoons ranarum Tchacarof, 1963 in R. septentrionalis. The aquatic frogs generally showed a much higher prevalence of infection with blood parasites than the terrestrial frogs, toads and salamanders, which is suggestive of an aquatic vector. The leech Batracobdella picta Verrill, 1872, which was found on many of the aquatic frogs, is the most likely vector in the study area. Also, an increasing prevalence of parasites was noted with increasing sizes (ages) of Rana clamitans and R. catesbeiana suggesting that longer exposure to water makes these species more likely to acquire blood parasites. The presence of Trypanosoma ranarum in B. americanus appeared to coincide with their attainment of sexual maturity.     

Allozyme comparison of three Trypanosoma species (Kinetoplastida: Trypanosomatidae) of toads and frogs by starch-gel electrophoresis.

Six metabolic enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, isocitrate dehydrogenase, malate dehydrogenase, phosphoglucomutase, and purine nucleoside phosphorylase, from clonal isolates of 3 presumptive species of Trypanosoma (T. fallisi, T. ranarum, and T. rotatorium) from 3 anuran hosts (Bufo americanus, Rana clamitans, and Rana catesbeiana) were compared using starch-gel electrophoresis. Although bands were shared among the different zymodemes of isolates of the same host genus, low genetic polymorphism of the enzyme loci was observed with few apparent shared bands between samples isolated from frogs and toads. A distance value calculated between toad and frog trypanosome isolates suggests the likelihood of long-time separation of species. Cluster analysis based on overall similarity distinguished the trypanosomes of toads and frogs as separate taxa, suggesting that host specificity and observed morphological differences are consistent with heritable allozyme differences.     

6-Hydroxydopamine produces a weight increase in toads

A large increase in body weight occurred after injection of 6-hydroxydopamine (6-OHDA, 100 mg kg-1) into the iliac lymph sac of toads. The body weight increase was apparently due to retention of fluid and only occurred when toads could not avoid contact of their ventral skin with water. Lower doses of 6-OHDA (25 or 50 mg kg-1) did not cause a weight increase although they were sufficient to eliminate fluorescence from peripheral adrenergic nerves. A peripheral injection of 6-OHDA did not affect the appearance of central aminergic nerves. Removal of the pituitary and median eminence did not affect the weight increase. Although the primary action of 6-OHDA in upsetting water balance in toads has not been revealed by this study, it is clearly not related to the chemical sympathectomy of adrenergic nerves.     

Chemosensory function of salt and water transport by the amphibian skin

Solute and water transport mechanisms of anuran skin mediate chemosensory functions that permit evaluation of ionic and osmotic properties of hydration sources in a manner similar to taste receptors in the mammalian tongue. Histochemical observations demonstrated apparent connections between spinal nerve endings and epithelial cells of the skin and we used neural and behavioral responses as measures of coupling between transport and chemosensation. The inhibition of transcellular Na+ transport by amiloride partially reduced the neural response and the avoidance of hyperosmotic NaCl but not KCl solutions. Cetylpyridinium chloride (CPC) reduced the neural response to hyperosmotic salt solutions, suggesting a chemosensory role for vanilloid receptors in the skin. Avoidance of hyperosmotic salt solutions was reduced by impermeant anions suggesting paracellular conductance is important for chemosensation. The effects of blocking the transcellular and paracellular pathways was additive but did not eliminate the avoidance of osmotically unfavorable solutions by dehydrated toads. The timing of the neural response to deionized water was similar to the onset of water absorption behavior and increased blood flow to the pelvic skin. Water absorption from 50 mM NaCl was greater than from deionized water when toads were fully immersed, but not when contact was limited to the ventral surface.     

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.     

Comparative effects of angiotensin II on osmotic water permeability in the toad (Bufo arenarum)

The possible relationship between the renin-angiotensin system and water balance in the toad Bufo arenarum has been indirectly explored. A positive correlation was found between the hydrosmotic response of ventral pelvic toad skin to angiotensin II (A II) and some age indicators (body weight, snout-urostyle length or head width). A different hydrosmotic response for oxytocin and isoproterenol (but not for A II) was found between four cutaneous regions of toad body. We conclude that A II may not be directly involved in the regulation of water balance mediated by water absorption across the skin of Bufo arenarum toads.     

Phylogenetic position of the giant anuran trypanosomes Trypanosoma chattoni,Trypanosoma fallisi,Trypanosoma mega,Trypanosoma neveulemairei,and Trypanosoma ranarum inferred from 18S rRNA gene sequences

Phylogenetic relationships within the kinetoplastid flagellates were inferred from comparisons of small-subunit ribosomal RNA gene sequences. These included 5 new gene sequences, Trypanosoma fallisi (2,239 bp), Trypanosoma chattoni (2,180 bp), Trypanosoma mega (2,211 bp), Trypanosoma neveulemairei (2,197 bp), and Trypanosoma ranarum (2,203 bp). Trees produced using maximum-parsimony and distance-matrix methods (least-squares, neighbor-joining, and maximum-likelihood), supported by strong bootstrap and quartet-puzzle analyses, indicated that the trypanosomes are a monophyletic group that divides into 2 major lineages, the salivarian trypanosomes and the nonsalivarian trypanosomes. The nonsalivarian trypanosomes further divide into 2 lineages, 1 containing trypanosomes of birds, mammals, and reptiles and the other containing trypanosomes of fish, reptiles, and anurans. Among the giant trypanosomes, T. chattoni is clearly shown to be distantly related to all the other anuran trypanosome species. Trypanosoma mega is closely associated with T. fallisi and T. ranarum, whereas T. neveulemairei and Trypanosoma rotatorium are sister taxa. The branching order of the anuran trypanosomes suggests that some toad trypanosomes may have evolved by host switching from frogs to toads.     

Vascular aspects of water uptake mechanisms in the toad skin: perfusion, diffusion, confusion

Blood cell flow (BCF) in the water absorbing "seat patch" region of toad skin was measured with laser Doppler flow cytometry. BCF of dehydrated toads increased by a factor of 6-8 when water contact was made and declined gradually as toads rehydrated. Water absorption was initially stimulated and declined in parallel with BCF. Water absorption measured during the initial rehydration period did not correlate with BCF and hydrated toads injected with AVT increased water absorption without an increase in BCF indicating the lack of an obligate relation between blood flow and water absorption. Aquaporins 1-3 were characterized by RT-PCR analysis of seat patch skin. AQP 1 was localized in the endothelium of subepidermal capillaries and serves as a pathway for water absorption in series with the apical and basolateral membranes of the epithelium. Dehydrated toads rehydrated more rapidly from dilute NaCl solutions than from deionized water despite the reduced osmotic gradient. BCF of toads rehydrating on 50 mM NaCl was not different than on deionized water and blocking Na+ transport with 100 microM amiloride did not reduce water absorption from 50 mM NaCl. Thus, neither circulation nor solute coupling explains the greater absorption from dilute salt solutions. Rehydration from 10 mM CaCl2 was stimulated above that of DI water by a similar degree as with 50 mM NaCl suggesting the anion might control water permeability of the skin.     

The spinal nerves innervate putative chemosensory cells in the ventral skin of desert toads, Bufo alvarius

Toads normally obtain water by absorption across their skin from osmotically dilute sources. When hyperosmotic salt solutions are presented as a hydration source to dehydrated desert toads, they place the ventral skin onto the source but soon afterwards escape to avoid dehydration. The escape behavior coincides with neural excitation of the spinal nerves that innervate putative chemosensory cells in the ventral skin. In the present study, fluorescent dye translocated through the spinal nerves to those receptor cells in the epidermis was photoconverted in the presence of 3, 3'-diaminobenzidine tetrahydrochloride for electron-microscopic observation of the cells and associated nerve terminals. Most of the photoconverted cells were located in the deepest layer of the epidermis, with some being in more intermediate layers. No labeled cell was seen in the outermost layer of living cells. In desert toads, flask cells and Merkel cells are occasionally seen in the epidermis. An association of nerve fibers with these epidermal cells has been reported in some species of the anurans. In the present study, however, the cytological features of the photoconverted cells are neither reminiscent of flask cells nor Merkel cells, but are similar to those of surrounding epithelial cells in each layer of the epidermis. We hypothesize a sensory function for these cells, because they have a close association with nerve fibers and participate in the transepithelial transport of salts that must pass through all cell layers of the skin.     

The localisation of lead in the skin of light- and dark-adapted Xenopus laevis

Toads pretreated for 2 months on either a dark or a light background were then exposed to lead nitrate at 50 ppm lead for 21 days, the illumination regimes being maintained. Metal analysis of dorsal skin showed significantly higher lead levels (p less than 0.01) in dark-adapted toads. No precipitated lead deposits were observed at the ultrastructural level, necessitating X-ray microanalysis of sections containing melanophores, gland cells and general (non-melanophore) cytoplasm. Analysis showed the lead to be concentrated within the melanosomes of the melanophores, and to be significantly higher (p less than 0.01) in individual melanosomes of dark-adapted toads than in light adapted ones. Copper was also found to be concentrated in the melanosomes and was higher (p less than 0.01) in the melanosomes of the dark-adapted toads. The results are consistent with the known affinity of melanin for heavy metals and the documented increase in melanophore number under prolonged dark background regimes. Since all toads received the same lead exposure, the melanosome results give rise to speculation that higher melanin levels might occur in individual melanosomes of dark-adapted skin.     

Temperature and humidity alter prolactin receptor expression in the skin of toad (Bufo bankorensis and Bufo melanostictus)

Bufo bankorensis and Bufo melanostictus, the only two species of Bufonidae genus in Taiwan, live in habitats that differ in altitude and humidity. This study tested the hypothesis that prolactin receptor (PRLR) expression responds to environmental change. Western blot analysis showed that the PRLR protein was widely distributed in brain, lung, liver, kidney, dorsal skin and ventral skin of toads. The level PRLR protein was elevated in the dorsal skin of the two toad species treated with dry or wet conditions for 14 days. The increase in PRLR of dorsal skin in B. bankorensis was higher than that in B. melanostictus. This experimental result suggests that B. bankorensis secretes more mucus to reduce water evaporation from its thinner cuticle than B. melanostictus. The expression of PRLR protein was increased in the lung of B. bankorensis and decreased in the lung of B. melanostictus. Moreover, PRLR protein levels were increased in the kidneys in the two species toad, likely due to reduction in water lost through lung and urine. The two toad species were subjected to varying temperatures (25 degrees C, 15 degrees C and 10 degrees C) for 14 days. The lowest PRLR protein expression was observed at 10 degrees C. Comparison of the decreasing trend in PRLR protein levels demonstrated that the variation in B. bankorensis was significantly higher than that in B. melanostictus. Comparisons of variation in PRLR protein expression in the two species under different environments suggest that B. bankorensis is more adaptable to different environments than B. melanostictus.