Rana Dalmatina Research Articles

Spontaneous succession on spoil banks supports amphibian diversity and abundance

The ecological value and conservation potential of post-mining areas have been increasingly recognized by scientists and conservationists during recent decades. Especially valuable are sites left to spontaneous succession, which constitute habitats with high species diversity, or habitats that serve as refuges for threatened species. In contrast to several other taxa, there is a lack of such evidence for amphibians, despite the assumption that primary succession leads to a more suitable environment for amphibians than does technical reclamation. Therefore, we compared the effects of technical reclamation and spontaneous succession on amphibian presence, species richness, and abundance of the model species Rana dalmatina in technically reclaimed and unreclaimed sections of spoil banks in the Czech Republic's North Bohemian brown coal basin. We found that most recorded amphibian species, and R. dalmatina in particular, occurred predominantly within successional spoil bank sections. Apart from reclamation status, amphibians preferred partially vegetated ponds (5–75% vegetation cover) having gently sloping shores (<30°) and lower water conductivity. Mean species richness per pond (1.95 vs. 1.20), the proportion of ponds occupied by amphibians (88.5% vs. 69.4%), and the mean numbers of R. dalmatina clutches per pond (9.05 vs. 1.65) were significantly higher at unreclaimed sites compared to technically reclaimed sites. This study confirms the conservation value of post-mining sites for amphibians and evidences that sites left to spontaneous succession provide more suitable habitats for amphibians compared to technically reclaimed sites. Key habitat characteristics driving amphibian assemblages within post-mining sites are identified and guidelines for effective protection of amphibians in post-mining areas are proposed.

No personality without experience? A test on Rana dalmatina tadpoles.

While the number of studies reporting the presence of individual behavioral consistency (animal personality, behavioral syndrome) has boomed in the recent years, there is still much controversy about the proximate and ultimate mechanisms resulting in the phenomenon. For instance, direct environmental effects during ontogeny (phenotypic plasticity) as the proximate mechanism behind the emergence of consistent individual differences in behavior are usually overlooked compared to environmental effects operating across generations (genetic adaptation). Here, we tested the effects of sociality and perceived predation risk during ontogeny on the strength of behavioral consistency in agile frog (Rana dalmatina) tadpoles in a factorial common garden experiment. Tadpoles reared alone and without predatory cues showed zero repeatability within (i.e., lack of personality) and zero correlation between (i.e., lack of syndrome) activity and risk-taking. On the other hand, cues from predators alone induced both activity and risk-taking personalities, while cues from predators and conspecifics together resulted in an activity - risk-taking behavioral syndrome. Our results show that individual experience has an unequivocal role in the emergence of behavioral consistency. In this particular case, the development of behavioral consistency was most likely the result of genotype×environment interactions, or with other words, individual variation in behavioral plasticity.

Immunodetection of telomerase-like immunoreactivity in normal and regenerating tail of amphibians suggests it is related to their regenerative capacity.

The high regenerative power of amphibians suggests that numerous signaling pathways remain active in larvae and adult tissues, including those stimulated by telomerases. The protein component of Telomerase (TERT) shows high amino acid homology in numerous vertebrates and we have utilized an antibody made for a lizard telomerase to detect a putative telomerase in amphibians tissues based on 60-75% epitope identity. Western blotting studies indicate that telomerase is constitutionally present in normal and regenerating tails of tadpoles of the anuran Rana dalmatina. In the neotenic urodele Ambystoma mexicanum the enzyme is detectable in the normal tail and increases during tail regeneration. Immunofluorescence shows that telomerase is localized in both epidermal and mesenchymal cells of normal and regenerating tail of A. mexicanum, especially in their nuclei. The ultrastructural localization with gold or gold-silver intensified confirms that telomerase is mainly present in the nucleus, in particular in discrete regions of the nucleoplasm identified as Cajal bodies and in the nucleolus, sites known in mammalian cells to be involved in the processing of the enzyme. Telomerases are present in normal and even more in regenerating tissues, and are possibly implicated in the numerous cell replications needed for physiological or post-amputation tissue regeneration in amphibians. J. Exp. Zool. 323A: 757-766, 2015. © 2015 Wiley Periodicals, Inc.

Choice of experimental venue matters in ecotoxicology studies: Comparison of a laboratory-based and an outdoor mesocosm experiment

The heavy application of pesticides and its potential effects on natural communities has attracted increasing attention to inadvertent impacts of these chemicals. Toxicologists conventionally use laboratory-based tests to assess lethal concentrations of pesticides. However, these tests often do not take into account indirect, interactive and long-term effects, and tend to ignore different rates of disintegration in the laboratory and under natural conditions. Our aim was to investigate the importance of the experimental venue for ecotoxicology tests. We reared tadpoles of the agile frog (Rana dalmatina) in the laboratory and in outdoor mesocosms and exposed them to three initial concentrations of a glyphosate-based herbicide (0, 2 and 6.5mg a.e./l glyphosate), and to the presence or absence of caged predators (dragonfly larvae). The type of experimental venue had a large effect on the outcome: The herbicide was less lethal to tadpoles reared in outdoor mesocosms than in the laboratory. Further, while the herbicide had a negative effect on development time and on body mass in the laboratory, tadpoles exposed to the herbicide in mesocosms were larger at metamorphosis and developed faster in comparison to those reared in the absence of the herbicide. The effect of the herbicide on morphological traits of tadpoles also differed between the two venues. Finally, in the presence of the herbicide, tadpoles tended to be more active and to stay closer to the bottom of laboratory containers, while tadpole behaviour shifted in the opposite direction in outdoor mesocosms. Our results demonstrate major discrepancies between results of a classic laboratory-based ecotoxicity test and outcomes of an experiment performed in outdoor mesocosms. Consequently, the use of standard laboratory tests may have to be reconsidered and their benefits carefully weighed against the difficulties of performing experiments under more natural conditions. Tests validating experimentally estimated impacts of herbicides under natural conditions and studies identifying key factors determining the applicability of experimental results are urgently needed.

Direct negative density‐dependence in a pond‐breeding frog population

Understanding population dynamics is critical for the management of animal populations. Comparatively little is known about the relative importance of endogenous (i.e. density‐dependent) and exogenous (i.e. density‐independent) factors on the population dynamics of amphibians with complex life cycles. We examined the potential effects of density‐dependent and ‐independent (i.e. climatic) factors on population dynamics by analyzing a 15‐yr time series data of the agile frog Rana dalmatina population from Târnava Mare Valley, Romania. We used two statistical models: 1) the partial rate correlation function to identify the feedback structure and the potential time lags in the time series data and 2) a Gompertz state‐space model to simultaneously investigate direct and delayed density dependence as well as climatic effects on population growth rate. We found evidence for direct negative density dependence, whereas delayed density dependence and climate did not show a strong influence on population growth rate. Here we demonstrated that direct density dependence rather than delayed density dependence or climate determined the dynamics of our study population. Our results confirm the findings of many experimental studies and suggest that density dependence may buffer amphibian populations against environmental stress. Consequently, it may not be easy to scale up from individual‐level effects to population‐level effects.

Behavioural consistency and life history of Rana dalmatina tadpoles.

The focus of evolutionary behavioural ecologists has recently turned towards understanding the causes and consequences of behavioural consistency, manifesting either as animal personality (consistency in a single behaviour) or behavioural syndrome (consistency across more behaviours). Behavioural type (mean individual behaviour) has been linked to life-history strategies, leading to the emergence of the integrated pace-of-life syndrome (POLS) theory. Using Rana dalmatina tadpoles as models, we tested if behavioural consistency and POLS could be detected during the early ontogenesis of this amphibian. We targeted two ontogenetic stages and measured activity, exploration and risk-taking in a common garden experiment, assessing both individual behavioural type and intra-individual behavioural variation. We observed that activity was consistent in all tadpoles, exploration only became consistent with advancing age and risk-taking only became consistent in tadpoles that had been tested, and thus disturbed, earlier. Only previously tested tadpoles showed trends indicative of behavioural syndromes. We found an activity-age at metamorphosis POLS in the previously untested tadpoles irrespective of age. Relative growth rate correlated positively with the intra-individual variation of activity of the previously untested older tadpoles. In previously tested older tadpoles, intra-individual variation of exploration correlated negatively and intra-individual variation of risk-taking correlated positively with relative growth rate. We provide evidence for behavioural consistency and POLS in predator- and conspecific-naive tadpoles. Intra-individual behavioural variation was also correlated to life history, suggesting its relevance for the POLS theory. The strong effect of moderate disturbance related to standard behavioural testing on later behaviour draws attention to the pitfalls embedded in repeated testing.

Fear is the mother of invention: anuran embryos exposed to predator cues alter life-history traits, post-hatching behaviour and neuronal activity patterns.

Neurophysiological modifications associated to phenotypic plasticity in response to predators are largely unexplored, and there is a gap of knowledge on how the information encoded in predator cues is processed by prey sensory systems. To explore these issues, we exposed Rana dalmatina embryos to dragonfly chemical cues (kairomones) up to hatching. At different times after hatching (up to 40 days), we recorded morphology and anti-predator behaviour of tadpoles from control and kairomone-treated embryo groups as well as their neural olfactory responses, by recording the activity of their mitral neurons before and after exposure to a kairomone solution. Treated embryos hatched later and hatchlings were smaller than control siblings. In addition, the tadpoles from the treated group showed a stronger anti-predator response than controls at 10 days (but not at 30 days) post-hatching, though the intensity of the contextual response to the kairomone stimulus did not differ between the two groups. Baseline neuronal activity at 30 days post-hatching, as assessed by the frequency of spontaneous excitatory postsynaptic events and by the firing rate of mitral cells, was higher among tadpoles from the treated versus the control embryo groups. At the same time, neuronal activity showed a stronger increase among tadpoles from the treated versus the control group after a local kairomone perfusion. Hence, a different contextual plasticity between treatments at the neuronal level was not mirrored by the anti-predator behavioural response. In conclusion, our experiments demonstrate ontogenetic plasticity in tadpole neuronal activity after embryonic exposure to predator cues, corroborating the evidence that early-life experience contributes to shaping the phenotype at later life stages.

Clutch Size Variation in Agile FrogRana dalmatinaon Post-Mining Areas

Clutch size is an important life history trait in amphibians, and it varies among and within species, populations and individuals. Within a population, its variation has been attributed to a positive relationship between females' age or size and their fecundity as well as to spatiotemporal differences in environmental conditions. Therefore, clutch size has been shown to be both spatially and seasonally variable. We examined spatial and seasonal clutch size variation based upon two years of study involving 160 clutches of the Agile Frog Rana dalmatina Bonaparte, 1840 in 14 ponds within one spoil bank in the Czech Republic's North Bohemian brown coal basin. The overall mean clutch size was 1295 (SD 596), which is one of the largest that has been reported. However, both clutch size and its variance differed considerably between the years. Clutch size also varied among the ponds. We found no relationship between clutch size and the distance of a breeding pond from alluvial forest, a typical wintering habitat. Despite existence at the site of many suitable reproduction habitats, the spoil bank does not offer the complex of all habitats needed for persistence of the R. dalmatina population. To protect that population, it is necessary to preserve not only breeding ponds on the spoil bank but also alluvial forest and, most importantly, the connectivity between these two crucial habitats.

Male calling activity in syntopic populations of Rana latastei and Rana dalmatina (Amphibia: Anura)

Rana latastei and Rana dalmatina are two explosive breeder amphibians whose mating seasons last less than 1 month. The two species have very different repertoires of vocalisations, as Rana dalmatina uses only one advertisement call while Rana latastei uses two different vocalisations with opposite structural features. In our research, we continually recorded the calling activity of the two species during a whole breeding season in a pond where they breed in syntopy in order to assess the possible functions of their vocalisations. Males of both species increased their activity in coincidence with the peak of activity of females, but Rana latastei males reached the peak 2–3 days before the deposition peak. By contrast, males of Rana dalmatina reached the peak at the same time as the deposition peak, and called at high intensity long after the deposition peak. These different acoustic patterns led us to infer different prevailing functions for the vocalisations of these species. Male vocalisations of Rana latastei are addressed only to males in order to gain and confirm their position within male hierarchy. By contrast, Rana dalmatina males might vocalise also in order to attract females, rather than to deter rival males. Thus, vocalisation in this second species might also play an intersexual function. The two call types of Rana latastei were used since the beginning of the breeding season. No difference was found also in daily activity, and both vocalisations were used preferentially during night time.

Chytridiomycosis risk among Central European amphibians based on surveillance data.

The Czech Republic hosts a surprisingly rich biodiversity of amphibians representing the majority of amphibian species present in all of Central and Eastern Europe. Surveillance data of Batrachochytrium dendrobatidis (Bd) collected during 2008 to 2012 were analysed for basic patterns of prevalence and infection intensity among species, age groups and localities. In addition, an investigation was made into possible data bias due to varying PCR inhibition. Infection prevalence in the genus Pelophylax was significantly higher than in other sampled taxa, while Bombina and Bufo were infected with intermediate prevalence. Individual mortalities putatively caused by chytridiomycosis were detected in Bombina and Bufo, but not in Pelophylax. Differences among localities were seen to modulate the pathogen's infection rate and influence overall individual infection intensities. PCR inhibition occurred significantly more often in samples from the genus Pelophylax than in other tested taxa (Bufo bufo, B. viridis, Bombina bombina, Pelobates fuscus and Rana dalmatina). Although we found no completely inhibited samples within the genus Bombina, the infection loads were lower in the sample set processed without bovine serum albumin, suggesting some level of PCR inhibition. The combination of high Bd prevalence with no apparent deleterious effect and the high dispersal abilities of water frogs predispose them to act as vectors for chytridiomycosis. It is possible that the role of Pelophylax frogs in the spread of Bd is overlooked due to a large proportion of unrecognized false negatives, but this issue needs further confirmation.

Reproductive interference between Rana dalmatina and Rana temporaria affects reproductive success in natural populations.

Experimental evidence suggests that reproductive interference between heterospecifics can seriously affect individual fitness; support from field studies for such an effect has, however, remained scarce. We studied reproductive interference in 25 natural breeding ponds in an area where two ranid frogs, Rana dalmatina and Rana temporaria, co-occur. The breeding seasons of the two species usually overlap and males of both species are often found in amplexus with heterospecific females, even though matings between heterospecifics produce no viable offspring. We estimated species abundance ratios based on the number of clutches laid and evaluated fertilization success. In ponds with low spatial complexity and a species abundance ratio biased towards R. temporaria, the average fertilization success of R. dalmatina eggs decreased, while this relationship was not detectable in spatially more complex ponds. Fertilization success of R. temporaria did not decrease with increasing relative numbers of heterospecifics. This asymmetry in fitness effects of reproductive interference may be attributed to R. temporaria males being more competitive in scramble competition for females than R. dalmatina males. Our study is among the first to demonstrate that in natural breeding populations of vertebrates interference among heterospecifics has the potential to substantially lower reproductive success at the population level, which may in turn affect population dynamics.

Ultrastructure and localisation of late-sporogonic developmental stages of Sphaerospora ranae (Myxosporea: Sphaerosporidae)

Data on external ultrastructure of myxospores and internal ultrastructure of advanced pseudoplasmodia and myxospores of topotypic samples of Sphaerospora ranae (Morelle, 1929) from Rana dalmatina Bonaparte are provided, together with in situ hybridisation results. In both frogs examined, the infection was restricted to renal tubules and corpuscles. The infection site restriction was confirmed by light and transmission electron microscopy, as well as by in situ hybridisation. In addition, large myxospore masses measuring up to 500 microm were detected in seminal vesicles. Only late-sporogonic stages, i.e. pseudoplasmodia harbouring immature and/or mature myxospores, were observed and analysed. Scanning electron microscopy revealed that spores have smooth surface with exception of posterior valvular bulges, which possess numerous outwards opening internal canals. As revealed by both scanning and transmission electron microscopy, the canals are continuous invaginations of the outer spore surface. Myxospores of S. ranae are characterised by the presence of two uninucleate sporoplasms, bilayered polar capsules, S/H-shaped polar filaments in transversal section and multilayered polar filament eversion pole plugging complex. Ultrastructural observations are discussed in the context of available data for other species of Sphaerospora sensu stricto and apparent synchronisation of myxospore shedding with a brief aquatic breeding phase of vertebrate intermediate host is highlighted.

What triggers the rising of an intraspecific biodiversity hotspot? Hints from the agile frog.

Hotspots of genetic diversity are regions of utmost importance for species survival and conservation, and their intimate link with the geographic location of glacial refugia has been well established. Nonetheless, the microevolutionary processes underlying the generation of hotspots in such regions have only recently become a fervent field of research. We investigated the phylogeographic and population genetic structure of the agile frog, Rana dalmatina, within its putative refugium in peninsular Italy. We found this region to harbour far more diversity, phylogeographic structure, and lineages of ancient origin than that by the rest of the species' range in Europe. This pattern appeared to be well explained by climate-driven microevolutionary processes that occurred during both glacial and interglacial epochs. Therefore, the inferred evolutionary history of R. dalmatina in Italy supports a view of glacial refugia as ‘factories' rather than as repositories of genetic diversity, with significant implications for conservation strategies for hotspots.

Contrasted influences of moon phases on the reproduction and movement patterns of four amphibian species inhabiting different habitats in central Italy

Many studies have provided evidence that prey adjust their behaviour to adaptively balance the fitness effects of reproduction and predation risk. Nocturnal terrestrial animals should deal with a range of environmental conditions during the reproductive season at the breeding sites, including a variable amount of natural ambient light. High degrees of illumination are expected to minimize those behaviours that might increase the animal detection by predators. Therefore, under habitat variable brightness conditions and in different ecosystems, the above mentioned behaviours are expected to depend on the variation in predation risk. Although moon effects on amphibian biology have been recognized, the direction of this influence is rather controversial with evidences of both increased and depressed activity under full moon. We tested in four nocturnal amphibian species (Hyla intermedia, Rana dalmatina, Rana italica, Salamandrina perspicillata) the effects of different (i) light conditions and (ii) habitats (open land vs. dense forest) on the reproductive phenology. Our results showed that the effects of the lunar cycle on the study species are associated with the change in luminosity, and there is no evidence of an endogenous rhythm controlled by biological clocks. The habitat type conditioned the amphibian reproductive strategy in relation to moon phases. Open habitat breeders (e.g., ponds with no canopy cover) strongly avoided conditions with high brightness, whereas forest habitat breeders were apparently unaffected by the different moon phases. Indeed, for all the studied species no effects of the moon phase itself on the considered metrics were found. Rather, the considered amphibian species seem to be conditioned mainly by moonlight irrespective of the moon phase. The two anurans spawning in open habitat apparently adjust their oviposition timing by balancing the fitness effects of the risk to be detected by predators and the reproduction.

Evolutionary trend for metamery reduction and gonad shortening in Anurans revealed by comparison of gonad development.

The gonads develop as the metameric mesodermal structures at the ventral surface of the mesonephroi. To study the evolutionary trends for anuran gonads, we performed comparative analysis of three species from the basal paraphyletic group - Archaeobatrachia (Bombina bombina, Xenopus laevis, Pelobates fuscus) and five species from more derived monophyletic Neobatrachia group (Hyla arborea, Bufotes viridis, Rana dalmatina, Rana arvalis, Rana temporaria). Light and scanning electron microscopy analysis showed that in Archaeobtrachia the undifferentiated gonads and ovaries, but not the testes, have pronounced external metamery. In contrast, Neobatrachia lacked external gonadal metamery and the internal metamery was well pronounced in the ovaries, but only rudimentary in the testes. There was also a difference in the site of genital ridge formation between these two groups. In Archaeobatrachia, the genital ridges developed along almost the entire length of mesonephroi, while in Neobatrachia they were limited to the anterior parts of the mesonephroi. A transient form was observed in P. fuscus, where the gonads formed along the anterior half of the mesonephroi. There was also an evolutionary trend for gradual postero-anterior shortening of the gonadal anlagen. In summary, our study indicates that among anurans there is an evolutionary trend for the gradual loss of metamery and shortening of the gonad.

New data on the distribution of the Italian agile frog Rana latastei Boulenger, 1879 in Slovenian Istra

After the first confirmed record of the Italian agile frog in the Dragonja River Valley, a herpetological field survey was organized in 2012 to estimate the species range in this area. We confirmed its reproduction in the Slovenian part of the watersheds of the rivers Dragonja, Mirna and upper part of Rižana. Here we present a preliminary report on the presence of this amphibian in the Slovenian part of Istra.

Egg Size Versus Clutch Size: Variation and Trade-off in Reproductive Output of Rana dalmatina and R. temporaria in a Pond near Bonn (Germany)

The common frog (R. temporaria) is a widespread and abundant species of woodland and agricultural landscapes in Europe and north-west Asia. In contrast, the distribution of the agile frog (R. dalmatina) is rather scattered and restricted mainly to deciduous and mixed forests in western, central and south-eastern Europe. Within their overlapping range, both species often breed in the same ponds. Although ecology of these brown frog species is well known in parts, many aspects of their biology possibly explaining niche differentiation and large and small scale distribution remain uncertain. Interspecific variation in reproductive output may be one key factor explaining local differences in their abundance and dispersal ability. Our study compares the reproductive output of both species in a single pond near Bonn (Germany) using data on egg size (diameter, egg mass), clutch size, body size, age, and body condition.

Better in the dark: two Mediterranean amphibians synchronize reproduction with moonlit nights

Abstract. In Amphibians, both positive and negative correlations between activity and full moon phase have been observed. In this study, we present data for two anuran species (Hyla intermedia and Rana dalmatina) studied in a hilly Mediterranean area of central Italy. We analysed, in a two-year survey, the relationships between the number of egg clutches laid each night and the moon phases by means of circular statistics. Moreover, the studied species exhibited clear oviposition site selection behaviour influenced, at least in H. intermedia, by moon phases. We observed the occurrence of an avoidance effect by amphibians for oviposition and specific egg-laying behaviour during moon phases around the full moon. This apparent lunar phobia was evident in both species when yearly data were pooled. On the other hand, while this pattern continued to be also evident in H. intermedia when single years were considered, in R. dalmatina it stood just in one year of study. Nonetheless, during cloudy nights, when moonlight arriving on the ground was low, the frogs' behaviour was similar to that observed in new moon phases. We interpreted the observed pattern as an anti-predatory strategy. Overall, comparisons between our own study and previous research suggest that there was insufficient evidence to establish any unequivocal patterns and that further research in this regard is needed.

A Tolna-Baranyai-dombvidék és a Kelet-Mecsek vizeinek kétéltű-faunisztikai vizsgálata

Between March and June 2012, we studied the presence of amphibian species in more than 100 waterbodies of the Tolna-Baranya Hills and the Eastern Mecsek. We included lakes, ponds, temporary pools, marshes as well as streams in our survey and considered all developmental stages of the inhabiting species. We found nine amphibian species in the whole territory. The two most common species were the water frog complex (Pelophylax esculentus complex) and the agile frog (Rana dalmatina). One of our most important results was proving the occurrence of yellow-bellied toad (Bombina variegata) in the Szekszárd Hills. The amphibian fauna was lesser-known in this region, thus our results are important for both biogeography and nature conservation.

Endosulfan Effects on Rana dalmatina Tadpoles: Quantitative Developmental and Behavioural Analysis

Endosulfan is an organochlorine pesticide that was recently labeled as a persistent organic pollutant, but it is still widely employed, particularly in developing countries. The goal of this study is to evaluate the acute (LC(50)) and chronic effects (developmental and behavioural traits) of this insecticide on Rana dalmatina tadpoles after exposure to ecologically relevant concentrations (0.005, 0.01, and 0.05mg/L) by applying video-tracking techniques to evaluate the quantitative effect of endosulfan on amphibian behavioural patterns. The 96h LC(50) value was 0.074mg endosulfan/L. Tadpoles chronically exposed to 0.01 and 0.05mg endosulfan/L underwent high mortality rate, decreased larval growth, delayed development, and increased incidence of malformations, and they did not reach metamorphosis by the end of the experiment. Moreover, tadpoles exposed to these concentrations exhibited several abnormalities in swimming patterns, such as shorter distance moved, swirling, resting, and unusual use of space. The exposure to 0.005mg endosulfan/L did not cause any significant effects on behaviour, larval growth, or development, but we observed a significant decrease in both survival and time to metamorphosis. We showed that developmental abnormalities are dose-dependent and that the pesticide effects could differ depending on the endosulfan concentration and the species tested. We also validated the hypothesis that behavioural analysis, along with the use of new analytical methods, could be a useful tool in amphibian ecotoxicological studies.