A light and electron microscopic study of Trypanosoma fallisi N. Sp. in toads (Bufo americanus) from Algonquin Park, Ontario

Trypanosoma fallisi n. sp. is described from Bufo americanus in Ontario. The parasite was observed in 65 of 94 toads examined. The trypanosomes were pleomorphic with respect to the age of infections, being longer and broader in early infections (during spring and summer) and shorter and more slender during late summer and autumn. They ranged in size from 38-76 microns in body length and 3-8 microns in width, with a free flagellum 6-30 microns long. Epizootiological and experimental evidence suggests that this trypanosome is transmitted to the toads by the leech, Batracobdella picta. Trypanosoma fallisi is morphologically similar to T. bufophlebotomi described in Bufo boreas from California, but geographic isolation, host and vector differences as well as slight morphological differences indicate that speciation has occurred. Similar trypanosomes from Bufo americanus (which were identified as T. bufophlebotomi) in Michigan, are probably T. fallisi. This species shares many ultrastructural features with trypanosomes of other lower vertebrates and also of mammals.     

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.     

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.     

A Light and Electron Microscopic Study of Trypanosoma fallisi N. Sp. in Toads (Bufo americanus) from Algonquin Park, Ontario

Trypanosoma fallisi n. sp. is described from Bufo americanus in Ontario. The parasite was observed in 65 of 94 toads examined. The trypanosomes were pleomorphic with respect to the age of infections, being longer and broader in early infections (during spring and summer) and shorter and more slender during late summer and autumn. They ranged in size from 38–76 μm in body length and 3–8 μm in width, with a free flagellum 6–30 μm long. Epizootiological and experimental evidence suggests that this trypanosome is transmitted to the toads by the leech, Batracobdella picta. Trypanosoma fallisi is morphologically similar to T. bufophlebotomi described in Bufo boreas from California, but geographic isolation, host and vector differences as well as slight morphological differences indicate that speciation has occurred. Similar trypanosomes from Bufo americanus (which were identified as T. bufophlebotomi) in Michigan, are probably T. fallisi. This species shares many ultrastructual features with trypanosomes of other lower vertebrates and also of mammals.     

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.     

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.     

Phototactic responses of anuran amphibians to monochromatic stimuli of equal quantum intensity

The phototactic responses of anuran amphibians to narrow-band monochromatic stimuli of equal quantum intensity were measured for the first time in eight new experiments. The unimodal spectral response, obtained from dark-adapted American toads (Bufo americanus), peaks near 626 THz of frequency (480 nm wavelength). The bimodal, U-shaped spectral response, obtained from dark-adapted tailed frogs (Ascaphus truei), has the anti-mode at about 589 THz (510 nm) and is not merely the spectral mirror-image of the unimodal response. Absolute level of the spectral stimuli of equal quantum intensity did not affect the spectral response of dark-adapted toads, but light-adaptation enhanced a component that has the same spectral peak as the visual pigment absorption spectrum of principal and single cones of the frog's retina.     

In vivo generation of 5-lipoxygenase products in frogs and toads

Eicosanoid production by inflammatory cells which resulted from infection of the peritoneal cavity of Rana catesbeiana and Bufo americanus was studied after addition of exogenous arachidonic acid and for metabolites generated in vivo. From exogenous substrate, the cells of Rana catesbeiana produced substantial amounts of 5-hydroxyeicosatetraenoic acid, leukotriene B4, the non-enzymatic isomers of leukotriene B4 and leukotriene C4. From endogenous substrate, 5-hydroxyeicosatetraenoic acid and leukotriene B4 were produced. Cells from Bufo americanus produced leukotriene B4 and 5-hydroxyeicosatetraenoic acid, from both exogenous and endogenous substrate. These observations of in vivo eicosanoid production confirm the participation of 5-lipoxygenase activity in the inflammatory response to infection.     

Helminth community structure of sympatric eastern American toad, Bufo americanus americanus, northern leopard frog, Rana pipiens, and blue-spotted salamander, Ambystoma laterale, from southeastern Wisconsin

One-hundred twelve amphibians, including 51 blue-spotted salamanders, Ambystoma laterale, 30 eastern American toads, Bufo americanus americanus, and 31 northern leopard frogs, Rana pipiens, were collected during April-October 1996 from Waukesha County, Wisconsin and examined for helminth parasites. The helminth compound community of this amphibian assemblage consisted of at least 10 species: 9 in American toads, 8 in leopard frogs, and 3 in blue-spotted salamanders. American toads shared 7 species with leopard frogs, and 2 species occurred in all 3 host species. Although there was a high degree of helminth species overlap among these sympatric amphibians, statistically significant differences were found among host species and percent of indirect or direct-life cycle parasites of amphibian species individual component communities (chi2 = 1,015, P < 0.001). American toads had a higher relative abundance of nematodes, 59%, than larval cestodes, 31%, and larval and adult trematodes, 10%, whereas leopard frogs had a higher relative abundance of larval cestodes, 71.3%, and larval and adult trematodes, 25.3%, than nematodes 3.4%. This is related to ecological differences in habitat and dietary preferences between these 2 anuran species. Helminth communities of blue-spotted salamanders were depauperate and were dominated by larval trematodes, 94%, and few nematodes, 6%. Low helminth species richness in this host species is related to this salamander's relatively small host body size, smaller gape size, lower vagility, and more fossorial habitat preference than the other 2 anuran species. Adult leopard frogs and toads had significantly higher mean helminth species richness than metamorphs, but there was no significant difference in mean helminth species richness among adult and metamorph blue-spotted salamanders. Considering adult helminths, the low species richness and low vagility of caudatans as compared with anurans suggest that local factors may be more important in structuring caudatan helminth communities of salamanders than of anuran hosts. Helminth species infecting salamanders may be more clumped in their geographic distribution as compared with anurans, and the role of other hosts and their parasites at the compound community level may be important in structuring helminth communities of salamanders.     

Direct and indirect effects of the introduced toad Bufo marinus (Anura: Bufonidae) on populations of native anuran larvae in Australia

Introduced species are frequently believed to have adverse effects on native biota and ecosystems. However, much of our knowledge of the ecological impacts of introduced species is anecdotal, and the mechanisms controlling these effects are often poorly understood. I used replicated artificial pond experiments to investigate the impact of eggs and hatchlings of the introduced toad Bufo marinus on populations of native anuran larvae (Limnodynastes ornatus and Litoria rubella) in Australia. Bufo marinus eggs and hatchlings are highly toxic to predatory native tadpoles. Under naturalistic conditions, populations of predatory L. ornatus tadpoles experienced significantly reduced survival when exposed to Bufo eggs and hatchlings. The impact of Bufo on L. ornatus survival was positively correlated with Bufo density. However, the toxic effects of Bufo on L. ornatus indirectly facilitated the survival of later-breeding L. rubella by altering predator-prey interactions between L. ornatus and L. rubella. Limnodynastes ornatus tadpoles are voracious predators of L. rubella eggs and hatchlings. Consequently, the negative impact of Bufo on populations of L. ornatus tadpoles reduced the intensity of predation by L. ornatus tadpoles on L. rubella eggs and hatchlings, thereby increasing L. rubella survival. The results demonstrate that B. marinus plays an important role in structuring native larval anuran communities via direct and indirect mechanisms, and that Bufo may have both negative and positive effects on populations of native anuran larvae. As far as I am aware, these are the first quantitative data to demonstrate that introduced fauna may affect populations of native biota via toxic effects.     

An electrically mediated block to polyspermy in the primitive urodele Hynobius nebulosus and phylogenetic comparison with other amphibians

At fertilization, the egg of the primitive urodele, Hynobius nebulosus, produced a fertilization potential which rose from -12 to +47 mV. A similar activation potential was elicited by pricking with a needle, by applying A23187, or by electric shock. The potential change was mediated by an increased permeability to Cl-. Clamping the egg's membrane potential at +40 mV blocked fertilization, while clamping at +20 mV induced polyspermy. These results indicated the occurrence of an electrical polyspermy block, typical of anurans, but atypical of urodeles. Furthermore, Hynobius eggs fertilized by natural mating incorporated only one sperm nucleus, and experimentally polyspermic eggs underwent multipolar division. Accessory sperm did not degenerate in the egg cytoplasm, indicating lack of an intracellular polyspermy block. By comparison, fertilization of Bufo japonicus (anuran) was also voltage dependent, whereas that of Cynops pyrrhogaster (urodele) was voltage independent. Thus polyspermy prevention mechanisms in Hynobius closely resemble those of anuran amphibians and differ from those of higher urodeles.     

Diet of Physalaemus cf. cicada (Leptodactylidae) and Bufo granulosus (Bufonidae) in a semideciduous forest.

We determined the diet of the two most abundant anuran species which occur in the litter of a semideciduous forest (Len?óis, Bahia, Brazil), Physalaemus cf. cicada and Bufo granulosus in the dry and rainy seasons. Pitfall traps were used to collect anuran and invertebrate fauna, which showed the availability of prey in the environment. Physalaemus cf. cicada was present in both seasons and Bufo granulosus only in the rainy season. Both species fed mainly on Isoptera and Formicidae. However, there is a difference between the rainy and dry seasons concerning the diet of P. cf. cicada. During the rainy season P. cf. cicada consumed less Isoptera and more Formicidae than in the dry season. In the volumetric sense, Orthoptera was the most important alimentary category for P. cf. cicada and B. granulosus. The Jacobs electivity index indicated that Physalaemus cf. cicada and Bufo granulosus were specialists in Isoptera.     

Pepsin in the toad Bufo marinus

The macroscopic localization of pepsinogen in the upper gastrointestinal tract of the anuran Bufo marinus was studied by means of biochemical assay. The pH optimum of the anuran pepsin was determined to be 1.6. The effect of lowered ambient temperature on stored pepsinogen and proteolytic activity was studied. Both parameters were reduced. The results are extrapolated to speculate on the mechanisms of gastric brooding in gastric brooding frogs of the genus Rheobatrachus.     

Ammonia as a pheromone in anuran tadpoles

In laboratory experiments, conspecific excretions and ammonia solutions evoked avoidance reactions in tadpoles of three anuran species, Bufo bufo, Rana temporaria, and Rana arvalis. A differential sensitivity of ammonia chemoreception was determined for two anuran species. For Bufo bufo tadpoles, these characteristics at ammonia background concentration of 0.2 mg/l lied in the range 150 % < dI/I < 500 %, and for background of 0.4 mg/l the value lied in the range 400 % < dI/I < 500 %. For Rana temporaria tadpoles, differential threshold against ammonia background concentration of 0.15 mg/l was close to 200 % and against background ammonia concentration of 1.1 mg/l was close to 100 %. These results suggest that such sensitivity of both anurans is sufficient for using ammonia in intra- and interspecies communication.     

Age Determination of Frogs and Toads (Amphibia, Anura) from North-western Europe

Age was determined in three anuran species inhabiting a temperate oceanic climate ( Rana temporaria, Bufo calamita and Bufo bufo ) by means of the histological structure of the phalanges. Narrow, haematoxylinophilic lines were visible in these, and evidence from the examination of subadults and frog ( R. temporaria ) recaptures showed the lines to be annual. The validity of using the lines as age indicators was tested most extensively on natural frog populations and the effects of endosteal resorption were not found to be significant. The evidence for this conclusion was provided directly by recapture data and indirectly by an analysis of histological measurements made on the phalanges.     

Histochemical determination of muscle fiber types in locomotor muscles of anuran amphibians.

1. A histochemical study using myosin ATPase, succinate dehydrogenase and alpha-glycerophosphate dehydrogenase reactions and a morphometric analysis with image analyser, was carried out in sartorius and gastrocnemius muscles of two anuran species, Rana perezi and Bufo calamita, that show different locomotor activities. 2. Four types of muscle fiber were found. There were interspecific variations in their proportions, with a predominance of oxidative muscle fibers in Bufo calamita. 3. These results agree with those obtained previously for the metabolic profile of several tissues from both species and point to a clear metabolic basis for the differences in locomotor activities between these two species.     

Evaluation of tadpole mouthpart depigmentation as a diagnostic test for infection by Batrachochytrium dendrobatidis for four California anurans

The objective of this study was to evaluate the utility of gross morphologic examination of larval mouthpart defects as a diagnostic screening test to detect Batrachochytrium dendrobatidis infection in four California, USA, anuran species. We examined mouthparts of 2,034 tadpoles of Bufo boreas, Pseudacris regilla, and Rana catesbeiana collected in 2003 and 2004 and Bufo canorus collected in 2004. Data were recorded for three morphologic features: upper toothrows, lower toothrows, and combined jaw sheaths. Mouthpart defects were observed in all four species (n=757), but only two species were infected with B. dendrobatidis (n=84). Sensitivity and specificity of the mouthparts test were 76% and 58%, respectively. Forty-two percent of B. dendrobatidis-negative animals would have been designated positive based on mouthpart defects. Observed prevalence was 43%, and true prevalence was 3.0%. Tests of the null hypothesis using logistic regression analysis showed that anuran larval mouthpart defects were not associated with B. dendrobatidis infection whether mouthparts scores were tested by individual morphologic feature or in combination (P=0.37). We conclude that B. dendrobatidis infection and anuran larval mouthpart defects are two separate processes that may occur concurrently and that evaluation of tadpole oral morphology is neither an accurate nor a reliable diagnostic test for B. dendrobatidis infection for the four species tested.     

Histochemical demonstration of 5-hydroxytryptamine in poison glands of amphibian skin

Summary The aim of this project was to see whether 5-hydroxytryptamine (5-HT) believed to be present in the skin glands of anuran amphibians can be demonstrated histochemically. The Periodic acid-Schiff technique (P.A.S.) and three histochemical methods known to demonstrate 5-HT in enterochromaffin cells (Pontana's, diazonium and Schmorl's) were applied to the dorsal skin of three species—Xenopus laevis, Rana angolensis and Bufo regularis.Mucous glands were identified by their P.A.S. reactivity in all three species. Poison glands were identifiable in Bufo regularis and Xenopus laevis only. The secretory granules of the latter glands have strong positive reactions with all three histochemical techniques used.It is concluded that the poison glands of anuran amphibians contain 5-HT demonstrable by histochemical means.     

Anuran fauna from Salta city, Argentina

The loss and transformation of habitat by anthropogenic activities is one of the main causes of biodiversity decrease. Amphibians are declining all over the world and one of the causes of this decline is thought to be habitat reduction. However, there are anuran populations in highly disturbed habitats, like cities. This study presents the ecological baseline needed to understand anuran communities in urban environments, for Salta City. Argentina (ca. 502,000 inhabitants). The sample was stratified by environments defined by infrastructure and geographic location. The species were identified by visual encounter techniques and by auditory transect during the 2002-2003 reproductive period. Three families, seven genera and twelve species were recorded and the species richness decreased from city border to center. Similarity index values among geographic areas was higher East-West (94.7) than South-West (33.3). Bufo arenarum was the only species recorded in all environments. Hyla andina and Odontophrynus americanus had a restricted distribution and are here considered vulnerable. The border effect can explain some distribution pattern in these populations. A map of the potential distribution of recorded species for Salta is provided.     

Influence of environmental conditions on the distribution of Central Asian green toads with three ploidy levels

We studied the distribution of Palearctic green toads (Bufo viridis subgroup), an anuran species group with three ploidy levels, inhabiting the Central Asian Amudarya River drainage. Various approaches (one‐way, multivariate, components variance analyses and maximum entropy modelling) were used to estimate the effect of altitude, precipitation, temperature and land vegetation covers on the distribution of toads. It is usually assumed that polyploid species occur in regions with harsher climatic conditions (higher latitudes, elevations, etc.), but for the green toads complex, we revealed a more intricate situation. The diploid species (Bufo shaartusiensis and Bufo turanensis) inhabit the arid lowlands (from 44 to 789 m a.s.l.), while tetraploid Bufo pewzowi were recorded in mountainous regions (340–3492 m a.s.l.) with usually lower temperatures and higher precipitation rates than in the region inhabited by diploid species. The triploid species Bufo baturae was found in the Pamirs (Tajikistan) at the highest altitudes (2503–3859 m a.s.l.) under the harshest climatic conditions.