Relationships Between Chemical Defenses of Common Toad (Bufo bufo) Tadpoles and Bacterial Community Structure of their Natural Aquatic Habitat.

Many organisms synthesize secondary metabolites against natural enemies. However, to which environmental factors the production of these metabolites is adjusted to is poorly investigated in animals, especially so in vertebrates. Bufadienolides are steroidal compounds that are present in a wide range of plants and animals and, if present in large quantities, can provide protection against natural enemies, such as pathogens. In a correlative study involving 16 natural populations we investigated how variation in bufadienolide content of larval common toads (Bufo bufo) is associated with the bacterial community structure of their aquatic environment. We also evaluated pond size, macrovegetation cover, and the abundance of predators, conspecifics and other larval amphibians. We measured toxin content of tadpoles using HPLC-MS and determined the number of bufadienolide compounds (NBC) and the total quantity of bufadienolides (TBQ). AICc-based model selection revealed strong relationships of NBC and TBQ with bacterial community structure of the aquatic habitat as well as with the presence of conspecific tadpoles. The observed relationships may have arisen due to adaptation to local bacterial communities, phenotypic plasticity, differential biotransformation of toxin compounds by different bacterial communities, or a combination of these processes. Bacterial groups that contribute to among-population variation in toxin content remain to be pinpointed, but our study suggesting that toxin production may be influenced by the bacterial community of the environment represents an important step towards understanding the ecological and evolutionary processes leading to microbiota-mediated variation in skin toxin profiles of aquatic vertebrates.

Negative effects of parasitic lung nematodes on the fitness of a Neotropical toad (Rhinella horribilis).

Pathogens are increasingly implicated in amphibian declines but less is known about parasites and the role they play. We focused on a genus of nematodes (Rhabdias) that is widespread in amphibians and examined their genetic diversity, abundance (prevalence and intensity), and impact in a common toad (Rhinella horribilis) in Panama. Our molecular data show that toads were infected by at least four lineages of Rhabdias, most likely Rhabdias pseudosphaerocephala, and multiple lineages were present in the same geographic locality, the same host and even the same lung. Mean prevalence of infection per site was 63% and mean intensity of infection was 31 worms. There was a significant effect of host size on infection status in the wild: larger toads were more likely to be infected than were smaller conspecifics. Our experimental infections showed that toadlets that were penetrated by many infective Rhabdias larvae grew less than those who were penetrated by few larvae. Exposure to Rhabdias reduced toadlet locomotor performance (both sustained speed and endurance) but did not influence toadlet survival. The effects of Rhabdias infection on their host appear to be primarily sublethal, however, dose-dependent reduction in growth and an overall impaired locomotor performance still represents a significant reduction in host fitness.

Changes in Toxin Quantities Following Experimental Manipulation of Toxin Reserves in Bufo bufo Tadpoles.

Possessing toxins can contribute to an efficient defence against various threats in nature. However, we generally know little about the energy- and time-demands of developing toxicity in animals, which determines the efficiency of chemical defence and its trade-off with other risk-induced phenotypic responses. In this study we examined how immersion into norepinephrine solution inducing the release of stored toxins, administration of mild stress mimicking predator attack or simple handling during experimental procedure affected the quantity and number of toxin compounds present in common toad (Bufo bufo) tadpoles as compared to undisturbed control individuals, and investigated how fast toxin reserves were restored. We found that total bufadienolide quantity (TBQ) significantly decreased only in the norepinephrine treatment group immediately after treatment compared to the control, but this difference disappeared after 12 h; there were no consistent differences in TBQ between treatments at later samplings. Interestingly, in the norepinephrine treatment approximately half of the compounds characterized by >700 m/z values showed the same changes in time as TBQ, but several bufadienolides characterized by <600 m/z values showed the opposite pattern: they were present in higher quantities immediately after treatment. The number of bufadienolide compounds was not affected by any treatments, but was positively related to TBQ. Our study represents the first experimental evidence that toxin quantities returned to the original level following induced toxin release within a very short period of time in common toad tadpoles and provide additional insights into the physiological background of chemical defence in this model vertebrate species.

Toxic activity and protein identification from the parotoid gland secretion of the common toad Bufo bufo.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

Volunteer Conservation Action Data Reveals Large-Scale and Long-Term Negative Population Trends of a Widespread Amphibian, the Common Toad (Bufo bufo).

Rare and threatened species are the most frequent focus of conservation science and action. With the ongoing shift from single-species conservation towards the preservation of ecosystem services, there is a greater need to understand abundance trends of common species because declines in common species can disproportionately impact ecosystems function. We used volunteer-collected data in two European countries, the United Kingdom (UK) and Switzerland, since the 1970s to assess national and regional trends for one of Europe's most abundant amphibian species, the common toad (Bufo bufo). Millions of toads were moved by volunteers across roads during this period in an effort to protect them from road traffic. For Switzerland, we additionally estimated trends for the common frog (Rana temporaria), a similarly widespread and common amphibian species. We used state-space models to account for variability in detection and effort and included only populations with at least 5 years of data; 153 populations for the UK and 141 for Switzerland. Common toads declined continuously in each decade in both countries since the 1980s. Given the declines, this common species almost qualifies for International Union for the Conservation of Nature (IUCN) red-listing over this period despite volunteer conservation efforts. Reasons for the declines and wider impacts remain unknown. By contrast, common frog populations were stable or increasing in Switzerland, although there was evidence of declines after 2003. "Toads on Roads" schemes are vital citizen conservation action projects, and the data from such projects can be used for large scale trend estimations of widespread amphibians. We highlight the need for increased research into the status of common amphibian species in addition to conservation efforts focusing on rare and threatened species.

Proliferative events and apoptotic remodelling in retinal development of common toad (Bufo bufo).

Proliferation and apoptosis are fundamental processes in the development of the retina, and a proper balance of the two phenomena is crucial to correct development of the organ. Despite intense investigation in different vertebrates, only a few studies have analyzed the cell death and the cell division quantitatively in the same species during development. Here we studied the time course of apoptosis and proliferation in the retina of common toad, Bufo bufo, and discuss the findings in an evolutionary perspective. We found cells that were dividing first scattered throughout the retina, then, in later stages, proliferation was confined to the ciliary marginal zone. This pattern was confirmed by the expression of the proliferative marker PCNA. Both proliferation and apoptosis occurred in successive waves, and two apoptotic peaks were detected: one at premetamorphosis 1 and the second at prometamorphosis. PARP-1, a known molecular marker of apoptosis, was used to confirm the data obtained by counting pyknotic nuclei. In summary, proliferative and apoptotic waves display an inverse time-relationship through development, with apoptotic peaks coinciding with low proliferation phases. In a comparative perspective, amphibians follow a developmental pattern similar to other vertebrates, although with different timing.

Screening breeding sites of the common toad (Bufo bufo) in England and Wales for evidence of endocrine disrupting activity.

Anuran amphibians are often present in agricultural landscapes and may therefore be exposed to chemicals in surface waters used for breeding. We used passive accumulation devices (SPMD and POCIS) to sample contaminants from nine breeding sites of the Common toad (Bufo bufo) across England and Wales, measuring endocrine activity of the extracts in a recombinant yeast androgen screen (YAS) and yeast estrogen screen (YES) and an in vitro vitellogenin induction screen in primary culture of Xenopus laevis hepatocytes. We also assessed hatching, growth, survival, and development in caged larvae in situ, and sampled metamorphs for gonadal histopathology. None of the SPMD extracts exhibited estrogen receptor or androgen receptor agonist activity, while POCIS extracts from two sites in west-central England exhibited concentration-dependent androgenic activity in the YAS. Three sites exhibited significant estrogenic activity in both the YES and the Xenopus hepatocyte. Hatching rates varied widely among sites, but there was no consistent correlation between hatching rate and intensity of agricultural activity, predicted concentrations of agrochemicals, or endocrine activity measured in YES/YAS assays. While a small number of intersex individuals were observed, their incidence could not be associated with predicted pesticide exposure or endocrine activitity measured in the in vitro screens. There were no significant differences in sex ratio, as determined by gonadal histomorphology among the study sites, and no significant correlation was observed between proportion of males and predicted exposure to agrochemicals. However, a negative correlation did become apparent in later sampling periods between proportion of males and estrogenic activity of the POCIS sample, as measured in the YES. Our results suggest that larval and adult amphibians may be exposed to endocrine disrupting chemicals in breeding ponds, albeit at low concentrations, and that chemical contaminants other than plant protection products may contribute to endocrine activity of surface waters in the agricultural landscape.

Isocline analysis of competition predicts stable coexistence of two amphibians.

We investigated the interaction between larvae of two anuran amphibian species (Rana temporaria and Bufo bufo) to test models of two-species competition. The study had a response surface experimental design with four replicates, each consisting of 24 density combinations. Larval performance-and, by assumption, change in population size-was defined by a linear combination of survival, growth, and development. We fit six competition models from the literature and discovered that density dependence was strongly non-linear, with the highest support for the Hassel-Comins model. Rana temporaria was competitively superior to B. bufo; the impacts of both species on growth and development were about five- to tenfold greater than those on survival. Isocline analysis predicted a stable configuration, which agrees with the observation that these two species are syntopic in nature. The results of this study confirm competition theory by identifying a model structure that agrees with data and making predictions that are broadly supportive of the observations.

Assessing atrazine-induced toxicities in Bufo bufo gargarizans Cantor.

Atrazine (AZ), a widely used herbicide has drawn attentions for its potential impacts on amphibians. This study aims to investigate the toxicity of AZ in Bufo bufo gargarizans Cantor (B. bufo gargarizans), a species of toad commonly found in China and countries in East Asia. We treated tadpoles with 0.1, 1, 10 and 100 µg/L AZ for 85 days and examined related parameters. The results showed that the mortality of the toads in the treatment group increased dramatically in a U-shaped dose-response relationship. The hindlimb extension and metamorphosis rate of the toads were significantly inhibited by AZ at 10 and 100 µg/L. Under the same condition, there were significant progressive changes in the testicular structures. Moreover, we found that AZ has no significant effects on growth, sex ratios, gonadal morphology, forelimb emergence and histology in the ovaries. Our results support the idea that environmental contaminants including AZ may be relevant to global amphibian decline.

Phenotypic divergence of the common toad (Bufo bufo) along an altitudinal gradient: evidence for local adaptation.

Variation in the environment can induce different patterns of genetic and phenotypic differentiation among populations. Both neutral processes and selection can influence phenotypic differentiation. Altitudinal phenotypic variation is of particular interest in disentangling the interplay between neutral processes and selection in the dynamics of local adaptation processes but remains little explored. We conducted a common garden experiment to study the phenotypic divergence in larval life-history traits among nine populations of the common toad (Bufo bufo) along an altitudinal gradient in France. We further used correlation among population pairwise estimates of quantitative trait (QST) and neutral genetic divergence (FST from neutral microsatellite markers), as well as altitudinal difference, to estimate the relative role of divergent selection and neutral genetic processes in phenotypic divergence. We provided evidence for a neutral genetic differentiation resulting from both isolation by distance and difference in altitude. We found evidence for phenotypic divergence along the altitudinal gradient (faster development, lower growth rate and smaller metamorphic size). The correlation between pairwise QSTs-FSTs and altitude differences suggested that this phenotypic differentiation was most likely driven by altitude-mediated selection rather than by neutral genetic processes. Moreover, we found different divergence patterns for larval traits, suggesting that different selective agents may act on these traits and/or selection on one trait may constrain the evolution on another through genetic correlation. Our study highlighted the need to design more integrative studies on the common toad to unravel the underlying processes of phenotypic divergence and its selective agents in the context of environmental clines.

[Individual variation in the development of the common toad, Bufo bufo (Anura, Bufonidae): I. Timing of development and anomalies of external structure].

Parameters of development have been analyzed in all offspring (1633 ind.) from the same spawn of one pair of common toads under conditions of laboratory rearing, with natural elimination being reduced to a minimum. The results show that the range of developmental options for the species is much wider than it follows from the standard tables of development. "Background anomalies" (Kovalenko, 2003) in the external structure of developing larvae are described and their probable frequencies under nearly optimal conditions are indicated.

[Individual variation in the development of the common toad, Bufo bufo (Anura, Bufonidae): 2. Diagnostic characters of the axial skeleton].

Analysis of individual variation in diagnostic characters of the axial skeleton has been performed in all offspring (1633 ind.) from the same spawn of one pair of common toads under conditions of laboratory rearing, with natural elimination being reduced to a minimum. More than 50 skeletal anomalies have been recorded. Factual data are presented that characterize deviations from the species norm in the frequency of "background anomalies" (Kovalenko, 2003). Correlations between manifestations of different variants of character states are considered. The problem of norm and anomaly in individual variation is discussed.

[Individual variation in the development of the common toad, Bufo bufo (Anura, Bufonidae): 3. Limitations of individual variation and their causes].

The results of studies on the axial skeleton of the common toad (a model species) have been used to analyze factual limitations of individual variations. The results show that the states of the studied characters do not freely combine with each other but are subject to certain morphogenetic limitations. The causes of most these limitations have been revealed during the study. Classification of the main factors limiting individual variation in the course of development is presented.

[Specific features of the melanophore system in different color morphs of larvae of the common toad (Bufo bufo L.)].

In a natural pond among usual black larvae of the common toad (Bufo bufo L.), a few unusual individuals of red-olive coloring were found out. In both morphs we investigated the melanophores of skin using different methods. The ESR-spectrometric analysis has shown the absence of distinctions between morphs by the amount of melanin. Analysis of total preparations of skin has shown the presence of various kinds of melanophore cells both in the derma and in the epidermis. Among typical melanophores, essentially differing cells appeared (atypical cells). In black morph tadpoles, the number of all kinds of melanophores is significantly greater than in red-olive morphs. It is shown that dark coloring is connected with a considerable number of atypical cells in the epidermis imposed on a dense layer of typical dermal melanophores with dispersed melanin.

Cd, Cu, Zn, Se, and metallothioneins in two amphibians, Necturus maculosus (Amphibia, Caudata) and Bufo bufo (Amphibia, Anura).

The accumulation of cadmium, its affinity for metallothioneins (MTs), and its relation to copper, zinc, and selenium were investigated in the experimental mudpuppy Necturus maculosus and the common toad Bufo bufo captured in nature. Specimens of N. maculosus were exposed to waterborne Cd (85 µg/L) for up to 40 days. Exposure resulted in tissue-dependent accumulation of Cd in the order kidney, gills > intestine, liver, brain > pancreas, skin, spleen, and gonads. During the 40-day exposure, concentrations increased close to 1 µg/g in kidneys and gills (0.64-0.95 and 0.52-0.76; n = 4), whereas the levels stayed below 0.5 in liver (0.14-0.29; n = 4) and other organs. Cd exposure was accompanied by an increase of Zn and Cu in kidneys and Zn in skin, while a decrease of Cu was observed in muscles and skin. Cytosol metallothioneins (MTs) were detected as Cu,Zn-thioneins in liver and Zn,Cu-thioneins in gills and kidney, with the presence of Se in all cases. After exposure, Cd binding to MTs was clearly observed in cytosol of gills as Zn,Cu,Cd-thionein and in pellet extract of kidneys as Zn,Cu,Cd-thioneins. The results indicate low Cd storage in liver with almost undetectable Cd in liver MT fractions. In field trapped Bufo bufo (spring and autumn animals), Cd levels were followed in four organs and found to be in the order kidney > liver (0.56-5.0 µg/g >0.03-0.72 µg/g; n = 11, spring and autumn animals), with no detectable Cd in muscle and skin. At the tissue level, high positive correlations between Cd, Cu, and Se were found in liver (all r > 0.80; α = 0.05, n = 5), and between Cd and Se in kidney (r = 0.76; n = 5) of autumn animals, possibly connected with the storage of excess elements in biologically inert forms. In the liver of spring animals, having higher tissue level of Cd than autumn ones, part of the Cd was identified as Cu,Zn,Cd-thioneins with traces of Se. As both species are special in having liver Cu levels higher than Zn, the observed highly preferential Cd load in kidney seems reasonable. The relatively low Cd found in liver can be attributed to its excretion through bile and its inability to displace Cu from MTs. The associations of selenium observed with Cd and/or Cu (on the tissue and cell level) point to selenium involvement in the detoxification of excessive cadmium and copper through immobilization.

Effects of ammonium nitrate on larval survival and growth of four Iberian amphibians.

The purpose of this study was to understand the differences in tolerance to ammonium nitrate in four Iberian amphibians. Results showed a negative effect on amphibian survival and larvae total length. Three different tolerance groups on amphibian survival were established. The two first groups showed a higher reduction, around 90% and between 70 and 80% after 96 h exposition at the maximum concentration tested (180 mg NH4NO3/L), while the third group showed no mortality in any concentration tested. In addition, a reduction in larvae total length was also detected for all four species, with Bufo bufo as the most sensitive species.

Environmentally relevant concentrations of endosulfan impair development, metamorphosis and behaviour in Bufo bufo tadpoles.

Endosulfan is a widely used organochlorine pesticide with well-documented neurotoxic effects in both humans and laboratory animals (mammals and fish). Neurotoxicity has been implied also in amphibians after short-term exposure to endosulfan. Little is known about effects of chronic exposure of endosulfan in amphibians. Previously, we examined the short-term toxicity of endosulfan in common toad (Bufo bufo) tadpoles and determined the LC50 value to 0.43 mg/L. In the present study, we investigated the effects of endosulfan on B. bufo tadpoles after chronic exposure to ecologically relevant concentrations. Tadpoles were exposed in a static renewal test, from shortly after hatching (Gosner stage 25) to completed metamorphosis, to 0.01, 0.05 and 0.1mg endosulfan/L (nominal). The exposure period lasted 43-52 days. Mortality, larval growth (mass), development (reached Gosner stage at various times and deformities presence), metamorphosis and behaviour (swimming activity) were monitored regularly over the entire course of larval development. Our results show that 0.05 and 0.1mg endosulfan/L caused impaired behaviour, prolonged time to metamorphosis, increased incidences of mouth and skeletal malformations as well as mortality, and reduced body weight (observed also at 0.01 mg/L) in B. bufo tadpoles. Behavioural effects occurred at exposure day 4, before any other effects occurred, indicating a neurotoxic effect. Endosulfan levels found in groundwater and surface water range from 0.1 to 100 microg/L and after extraordinary runoff events, concentrations exceed 0.5 mg/L in surface water. Our results indicate that endosulfan may negatively affect wild frog populations in agricultural areas.

Morphological aspects of gonadal morphogenesis in Bufo bufo (Amphibia Anura): Bidder's organ differentiation.

We have described the architecture of Bidder's organ, defined its compartmented structure, and affirmed the presence of basal laminae. We did not find morphological differences between sexes in Bidder's organ. All specimens initially developed gonads with a peripheral fertile layer surrounding a thin primary cavity. The first oogenetic wave was observed early, showing all phases of meiosis, including leptotene, zygotene, and pachytene, which had been previously thought to be lacking. The peculiar presence of an asynchronous germ cell nest was discussed. Diplotene oocytes issued from the peripheral layer and migrated inside the primary cavity. They were surrounded by a single layer of follicular cells, which originated from the peripheral layer somatic cells and were delimited by a basal lamina. There were few medulla or central layer cells. At the end of metamorphosis, while the oocytes of the first oogenetic wave came into close contact with blood vessels, a second oogenetic wave took place just as the first, except for the presence of synchronous germ cell nests. The central layer was not visible and we did not observe the formation of an ovarian pocket. Stocks of stem germ cells remained in the peripheral layer during both the first and second oogenetic waves. The asymmetric model, in which there is a tendency toward a primary female differentiation, was confirmed. The female differentiation becomes stable in the Bidder's organ because of the absence of further interaction between germ and medullary somatic cells, which would have led toward a male differentiation.

Social aggregation and lateralised response to social stimuli in tadpoles (Bufo bufo): influence of developmental stage.

In previous studies several species of tadpoles showed a preference for using the left hemifield when approaching a conspecific and during fixation of their own mirror images. These lateral biases typically emerge 5 minutes after the placement of the animals in the test apparatus possibly due to variation in tadpoles' social propensity during the test. The aim of this study is to check whether lateralised response to social stimuli in tadpoles varies during their development. I compared tadpoles of three age classes from a few days after emergence from eggs to close to metamorphosis. In accordance with previous studies I found that tadpoles displayed a preference for the left hemifield when approaching a conspecific and that the bias emerged 5 minutes after placement in the test apparatus. No marked change with development was observed, yet the degree of laterality tended to decrease with age and tadpoles of intermediate age showed left-hemifield dominance during the test significantly earlier than the other two groups.