Rana Dalmatina Research Articles

Juvenile agile frogs spatially avoid ranavirus-infected conspecifics

Exposure to contagious pathogens can result in behavioural changes, which can alter the spread of infectious diseases. Healthy individuals can express generalized social distancing or avoid the sources of infection, while infected individuals can show passive or active self-isolation. Amphibians are globally threatened by contagious diseases, yet their behavioural responses to infections are scarcely known. We studied behavioural changes in agile frog (Rana dalmatina) juveniles upon exposure to a Ranavirus (Rv) using classic choice tests. We found that both non-infected and Rv-infected focal individuals spatially avoided infected conspecifics, while there were no signs of generalized social distancing, nor self-isolation. Avoidance of infected conspecifics may effectively hinder disease transmission, protecting non-infected individuals as well as preventing secondary infections in already infected individuals. On the other hand, the absence of self-isolation by infected individuals may facilitate it. Since infection status did not affect the time spent near conspecifics, it is unlikely that the pathogen manipulated host behaviour. More research is urgently needed to understand under what circumstances behavioural responses can help amphibians cope with infections, and how that affects disease dynamics in natural populations.

Comparative analysis of protein profiles in skin secretions of some Rana species: Preliminary insights into antimicrobial activity

Protein profiles of skin secretions of Rana dalmatina (Agile Frog), Rana macrocnemis (Uludağ Frog), Rana tavasensis (Tavas Frog) and Rana holtzi (Taurus Frog) frog species belonging to the Rana genus distributed in the Anatolian region of Türkiye were determined for the first time using the Tricine-SDS-PAGE Electrophoresis method and Coomassie Brilliant Blue (CBB) staining. By the results, some peptides with mass ≤5 kDa were detected. Just one peptide with mass ≤5 kDa was found in the secretion of each R. dalmatina, R. macrocnemis, and R. tavasensis while there was two in R. holtzi secretion. The antibacterial activity of secretions was determined using plate well diffusion assay on E. coli, S. typhimurium, S. aureus, B. cereus and L. monocytogenes bacteria. R. dalmatina created the inhibition zone for S. typhimurium, S. aureus, B. cereus, and L. monocytogenes. The zones of inhibition by R. tavasensis and R. macrocnemis species secretions were observed on S. aureus, B. cereus, and L. monocytogenes. It was found that R. holtzi creates an inhibition zone only on B. cereus. The results showed that the secretion of none of the species doesn't have antibacterial activity on E. coli. The skin secretion of R. dalmatina showed the most activity against bacteria, while R. holtzi had the least.

Lernaea cf. cyprinacea infection in agile frog (Rana dalmatina) tadpoles in Hungary.

The ectoparasitic copepod Lernaea cyprinacea (anchor worm) has more than 100 host species among teleost fishes and affects cyprinids both in fish farms and natural waters. In addition, while L. cyprinacea infection in amphibians has been recorded in Asia, North and South America, there is no data available in the literature on their presence in Europe. In this study, we first reported L. cf. cyprinacea parasitising an anuran tadpole in Europe. Specimens of L. cf. cyprinacea were observed attached to a tadpole of the agile frog (Rana dalmatina), which was caught during fishing of crucian carp fingerlings (Carassius carassius) from a small fish pond in Hungary during the summer of 2012. The infected tadpole was collected from a rearing pond, where juvenile crucian carps were kept. The tadpole was inspected in the laboratory, and digital photos were taken. The parasites were found attached to the body-tail junction and to the leg of the tadpole (at Gosner stage 41). The parasite species was identified as L. cf. cyprinacea based on morphological traits.

The ontogenetic development of Rana dalmatina Bonaparte 1840 species (Amphibia: Ranidae) in the context of climate change

Globally, amphibians represent one of the main objects of study in the context of climate change, because the temperature variations recorded and in the Republic of Moldova also represent a major threat to their development and sustainability. For this purpose, a complex ecological study was carried out regarding the embryonic and larval development strategies of the Rana dalmatina Bonaparte, 1840, species and the main particularities related to its population structure and dynamics were described. This research allowed us to evaluate the degree of ecological plasticity of natural populations of Rana dalmatina in relation to the plurality of environmental factors, which allows us to develop suitable and safe measures for monitoring and conservation of the species.

Epidermal cell proliferation and differentiation in the beak of tadpoles of Rana dalmatina

AbstractEpidermal cell proliferation and differentiation during formation of the beak in tadpoles of Rana dalmatina. Acta Zoologica (Stockolm). The beak utilized in tadpoles of R. dalmatina for crushing vegetables is formed of cornified cell layers. A row of large wedge‐like cells forms a central pile that produces the sharp corneous lamina of the beak. The labial and oral surfaces of the beaks also accumulate corneous material during proliferation and differentiation of keratinocytes from the epidermis to form a sheath around the central corneous cells. Corneocytes are shed during the growth of tadpoles. Electron microscopy reveals that keratin and mucus granules are main components of these keratinocytes. Electron‐dense organelles of unknown composition and with inside lamellae are also present. Using immunohistochemistry after post‐injection of 5BrdU, a marker of cell proliferation, the main timing of formation of the beak has been determined. After 2–5 h from injection, labelled nuclei of keratinocytes are seen in the basal layer, and sparse suprabasal labelled nuclei are detected after 5 h. Labelled keratinocytes are seen in suprabasal layers at 2 days post‐injection. At 5 and 6 days labelled keratinocytes are present in central corneous cells and in the lateral shell of corneocytes forming the labial and oral beak sides. Shedding a complete stratum corneum likely takes more days, but this depends on feeding usage.

Interspecific variation in the defensive responses of brown frogs to alien predators

AbstractAlien species impact native amphibians through various direct effects, including predation, and pose a significant threat to naïve prey populations. In this study, we exposed tadpoles of three brown frog species (Rana dalmatina, Rana latastei, and Rana temporaria) to the olfactory cues of two alien predators, the pond slider (Trachemys scripta) and red swamp crayfish (Procambarus clarkii), and compared their responses to those induced by the aquatic larvae of a widespread native predator, the Southern hawker (Aeshna cyanea). We recorded two tadpole defensive behaviors, the proportion of time they were active and the number of freezing events. Both agile frog species, R. dalmatina and R. latastei, showed moderate responses to red swamp crayfish kairomones and strong responses to both odonate larvae and pond sliders. In contrast, the common frog (R. temporaria) displayed a less intense response to crayfish with respect to odonate larvae, and a negligible response to pond sliders. Long-lasting coexistence with either European pond turtles or pond sliders may explain the strength of agile frogs’ response toward the alien species; while, the historical range of the white-clawed crayfish (Austropotamobius pallipes) overlapped that of the common frog, enhancing the co-option of alien crayfish cues by this anuran species.

Morphological and life-history plastic responses to predators and competitors in two brown frogs,Rana dalmatina and R. Latastei

Predation and competition are two major factors that drive natural selection. When they vary unpredictably, selection has promoted the evolution of plastic responses in behavioral, morphological and life-history traits. These responses are not independent of each other and often represent a trade-off between conflicting interests. We conducted a common-garden experiment to study the effects of predation and competition on the morphology and life history of R. dalmatina and R. latastei tadpoles. The experiment used a randomized-block design, where tadpoles were raised either with or without siblings and either with or without predators. Regardless of the treatments, R. dalmatina grew faster, developed proportionally larger tails, proportionally smaller bodies, and completed metamorphosis earlier than R. latastei. Both species developed relatively larger bodies with competitors, and relatively larger tails with predators. While the relative increase in body size with competitors was similar in the two species, the increase in tail size with predators was greater in R. dalmatina, suggesting that this species invested more in defense than R latastei. Competitors delayed metamorphosis in both species and in R. latastei they negatively affected froglet body size and shape. Conversely, predators delayed the metamorphosis only in R. dalmatina. This delay was the long-term cost paid by R. dalmatina for the short-term benefits of developing more effective behavioral and morphological defensive traits.

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

Contrasting effects of ammonium nitrate on tadpole survival, growth and behavior in two common anuran species

Extensive fertilizer use often leads to an accumulation of chemicals in waterbodies that are in close proximity to agricultural fields. One such commonly used fertilizer, ammonium nitrate, has been shown to negatively affect anuran survival and behavior. We performed experiments on tadpoles of two amphibian species, the Agile frog (Rana dalmatina) and the Common toad (Bufo bufo), to test the effects of different concentrations (0 mg/L, 50 mg/L, 100 mg/L and 200 mg/L) of ammonium nitrate on survival, growth and movement activity. We also examined whether sensitivity to predator cues might be altered in the presence of ammonium nitrate. Finally, we tested for the capacity of the two species to exhibit avoidance behavior. Overall, we found that the Agile frog is more sensitive to ammonium nitrate than the Common toad in terms of survival, growth and movement activity. Sensitivity to predator cues was not affected by the fertilizer for the Agile frog, but was shown to alter the behavior of Common toads, whose movement activity did not decrease in the presence of predator cues. Neither species showed an avoidance behavior. Our results suggest that ammonium nitrate can have significant effects on Agile frog tadpoles through direct toxicological effects on survival and growth, but also through behavioral effects. For Common toads those effects were less obvious, but were nonetheless shown in terms of growth, activity and predator detection.

Three brown frog species in Denmark have different abilities to colonise new ponds

For 29 consecutive years, the populations of three species of brown frogs, Rana arvalis, R. dalmatina and R. temporaria, were monitored in an open area in South Zealand, Denmark, with no direct influence of agriculture. Population sizes were recorded by counting egg clumps and showed large variations from year to year. The total population of R. arvalis differed by a factor of 100 between the years with the lowest and the highest numbers. A total of 19 initially unoccupied suitable waterbodies could potentially be colonised by the frogs. Rana dalmatina colonised all of the ponds, mostly in the very first year of existence. Rana arvalis colonised 17 ponds after an average of 10.5 years. Rana temporaria colonised eight ponds after an average of 13.4 years. Colonisation by R. dalmatina was independent of changes in total population size, whereas colonisation by R. arvalis predominantly occurred in years with considerable population increases. The results are discussed in relation to the movement patterns and philopatry of juvenile frogs of the three species. Juvenile R. dalmatina disperse far from the breeding site, but most individuals return to their natal site. This allows the species to be an efficient coloniser of new waterbodies and, at the same time, to have stable occurrence at the original site. The two other species show a more erratic type of dispersal and especially R. temporaria often shifts breeding site from year to year.

Tadpoles Develop Elevated Heat Tolerance in Urban Heat Islands Regardless of Sex

The ability of wildlife to endure the effects of high temperatures is increasingly important for biodiversity conservation under climate change and spreading urbanization. Organisms living in urban heat islands can have elevated heat tolerance via phenotypic or transgenerational plasticity or microevolution. However, the prevalence and mechanisms of such thermal adaptations are barely known in aquatic organisms. Furthermore, males and females can differ in heat tolerance, which may lead to sex-biased mortality, yet it is unknown how sex differences in thermal biology influence urban phenotypic divergence. To address these knowledge gaps, we measured critical thermal maxima (CTmax) in male and female agile frog (Rana dalmatina) tadpoles captured from warm urban ponds and cool woodland ponds, and in a common-garden experiment where embryos collected from both habitat types were raised in the laboratory. We found higher CTmax in urban-dwelling tadpoles compared to their counterparts living in woodland ponds. This difference was reversed in the common-garden experiment: tadpoles originating from urban ponds had lower CTmax than tadpoles originating from woodland ponds. We found no effect of sex on CTmax or its difference between habitats. These results demonstrate that aquatic amphibian larvae can respond to the urban heat island effect with increased heat tolerance similarly to other, mostly terrestrial taxa studied so far, and that phenotypic plasticity may be the main driver of this response. Our findings also suggest that heat-induced mortality may be independent of sex in tadpoles, but research is needed in many more taxa to explore potentially sex-dependent urban thermal responses.

New data regarding the distribution and status of the herpetofauna from urban and peri-urban habitats in the city of Pitești, Argeș county (Romania)

Identifying urban and peri-urban areas inhabited by species of reptiles and amphibians represents the first step in understanding how herpetofauna can adapt to anthropogenic factors. This is particularly true for regions where sampling biases have left significant gaps in our knowledge of fauna distribution, such as Romania. The aim of the study is to show my preliminary observations on the herpetofauna and its use of habitats in the urban and peri-urban environments from the city of Pitești, Argeș County, located in southern Romania. I identified nine species of amphibians (Salamandra salamandra, Lissotriton vulgaris, Triturus cristatus, Bombina variegata, Bufo bufo, Bufotes viridis, Hyla orientalis, Pelophylax ridibundus and Rana dalmatina) and nine species of reptiles (Emys orbicularis, Trachemys scripta, Lacerta agilis, Lacerta viridis, Podarcis muralis, Anguis colchica, Coronella austriaca, Natrix natrix and Natrix tessellata). The most abundent species in urban environments were Podarcis muralis and Bufotes viridis, with other species naturally present in the same habitats being Lacerta viridis, Hyla orientalis and Bufo bufo. In an artificial urban pond there were present Emys orbicularis and the invasive species Trachemys scripta (together with other exotic turtles belonging to other genera) as a result of translocation and releases. When taking into account the peri-urban areas as well, the most abundant species in the study area were Bombina variegata, Rana dalmatina and Bufotes viridis, the first two being limited to the woodland area. The most widespread species were Hyla orientalis, Bufotes viridis, Lacerta viridis, Podarcis muralis and Natrix natrix. These widespread species were present throughout all the study area. I have also recorded some species of reptiles with very few observations in the Argeș County: Natrix tessellata and Trachemys scripta. Finally, I identified anthropogenic factors that negatively impact the herpetofauna, such as habitat loss, direct persecution, road mortality, invasive species and poaching.

Does stress make males? An experiment on the role of glucocorticoids in anuran sex reversal.

Environmentally sensitive sex determination may help organisms adapt to environmental change but also makes them vulnerable to anthropogenic stressors, with diverse consequences for population dynamics and evolution. The mechanisms translating environmental stimuli to sex are controversial: although several fish experiments supported the mediator role of glucocorticoid hormones, results on some reptiles challenged it. We tested this hypothesis in amphibians by investigating the effect of corticosterone on sex determination in agile frogs (Rana dalmatina). This species is liable to environmental sex reversal whereby genetic females develop into phenotypic males. After exposing tadpoles during sex determination to waterborne corticosterone, the proportion of genetic females with testes or ovotestes increased from 11% to up to 32% at 3 out of 4 concentrations. These differences were not statistically significant except for the group treated with 10 nM corticosterone, and there was no monotonous dose-effect relationship. These findings suggest that corticosterone is unlikely to mediate sex reversal in frogs. Unexpectedly, animals originating from urban habitats had higher sex-reversal and corticosterone-release rates, reduced body mass and development speed, and lower survival compared to individuals collected from woodland habitats. Thus, anthropogenic environments may affect both sex and fitness, and the underlying mechanisms may vary across ectothermic vertebrates.

PRELIMINARY OBSERVATIONS REGARDING THE HERPETOLOGICAL FAUNA FROM CORCOVA (MEHEDINȚI COUNTY)

The paper presents preliminary data on amphibian and reptile fauna from Corcova (Mehedinți county). The commune, with an area of 75.32 km2, is located in the south-west of Romania (north-east of the county), on the right bank of the Motru River. Hills prevail in terms of landforms. The study aimed to update the herpetological database with new information resulting from research conducted between March and October 2022. The preliminary list includes: 10 species of amphibians (Urodela - 3 species: Salamandra salamandra, Triturus cristatus, Lissotriton vulgaris and Anura - 7 species: Bombina bombina, Hyla orientalis, Pelobates fuscus, Bufotes viridis, Rana dalmatina, Pelophylax kl. esculentus, P. ridibundus) and 11 species of reptiles (2 species of turtles: Testudo hermanni, Emys orbicularis; 4 species of lizards: Lacerta agilis, L. viridis, Podarcis tauricus, Anguis fragilis; 5 snake species: Dolichophis caspius, Coronella austriaca, Zamenis longissimus, Natrix natrix, N. tessellata). The variety of habitats, the temperate-continental climate with sub-Mediterranean influences, the trophic resource have influenced the diversity of herpetofauna in the area. The highlighted species of amphibians and reptiles are listed on different national protectionist lists (ordinances, laws, etc.). Species that are subject to special habitat conservation measures have drawn our attention: Testudo hermanni, Emys orbicularis, Triturus cristatus, Bombina bombina, Pelobates fuscus (Annex 3 to OUG 57/2007).

Tandem Mass Spectrometry de novo Sequencing of the Skin Defense Peptides of the Central Slovenian Agile Frog Rana dalmatina.

Peptides released on frogs' skin in a stress situation represent their only weapon against micro-organisms and predators. Every species and even population of frog possesses its own peptidome being appropriate for their habitat. Skin peptides are considered potential pharmaceuticals, while the whole peptidome may be treated as a taxonomic characteristic of each particular population. Continuing the studies on frog peptides, here we report the peptidome composition of the Central Slovenian agile frog Rana dalmatina population. The detection and top-down de novo sequencing of the corresponding peptides was conducted exclusively by tandem mass spectrometry without using any chemical derivatization procedures. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), electron transfer dissociation (ETD) and combined MS3 method EThcD with stepwise increase of HCD energy were used for that purpose. MS/MS revealed the whole sequence of the detected peptides including differentiation between isomeric Leu/Ile, and the sequence portion hidden in the disulfide cycle. The array of the discovered peptide families (brevinins 1 and 2, melittin-related peptides (MRPs), temporins and bradykinin-related peptides (BRPs)) is quite similar to that of R. temporaria. Since the genome of this frog remains unknown, the obtained results were compared with the recently published transcriptome of R. dalmatina.

Decline and Extinction of the Italian Agile Frog Rana latastei from Core Areas of Its Range.

Detecting the trends of species and populations is fundamental to identifying taxa with high conservation priority. Unfortunately, long-term monitoring programs are challenging and often lacking. The Italian agile frog Rana latastei is endemic to Northern Italy and adjacent countries, is considered vulnerable by the IUCN, and is protected at the European level. However, quantitative estimates of its decline are extremely scarce. In this study, we document the trends in abundance and distribution of Rana latastei within Monza Park, which currently represents the area closer to the type locality of the species and holds unique genetic features. Wetlands within the park were monitored from 2000 to 2023; counts of egg clutches were taken as a measure of reproductive output and the abundance of breeding females. In 2000, the species occurred over a significant proportion of the park. Total abundance showed strong yearly variation but remained rather constant from 2000 to 2019. However, Rana latastei disappeared from the park around 2021 and was never detected in 2022-2023. The decline is probably related to the joint effect of multiple factors, including the conversion of breeding sites for farming, inappropriate water management, invasive alien species, and severe drought. The local extinction of Rana latastei occurred despite legal protection, highlighting the need for more effective and stringent tools for the conservation of European biodiversity.

Anti-predator behavior in two brown frogs: differences in the mean behaviors and in the structure of animal personality variation

Predation is a major source of selection and prey are known to modify their behavior depending on their past experiences and the current perceived risk. Within a species, variation in experience and in the response to perceived risk combine to explain variation in personality and individual plasticity. Between species, variation in personality and plasticity might also be the evolutionary consequence of different selective regimes. In this study, we describe the anti-predator behavior of two closely related brown frogs, Rana dalmatina and Rana latastei, and compare their structures of personality variation. We raised tadpoles in a common garden experiment with either fish, dragonfly larvae, or no predators. Tadpoles were then repeatedly tested in the presence of the three acute stimuli and their behavioral variation was described in terms of quantity and quality of movements and of path sinuosity. In these tests, tadpoles of both species and ontogenetic treatments responded flexibly to predators by moving less, faster, and with more tortuous movements, and tadpoles raised with predators tended to move even faster. Independent of the acute treatment, R. dalmatina moved more and faster than R. latastei and the differences were larger without than with predators, demonstrating its higher plasticity. At the individual level, the two species showed qualitatively similar but quantitatively different structures of personality variation. R. dalmatina, more active, faster, and more plastic than R. latastei, showed also higher repeatability and a larger behavioral variation both among and within individuals.Significance statementPredators are a major source of selection and preys have evolved the ability to flexibly respond to them. These responses often vary among species, because of their different evolutionary histories, and among individuals, because of their different experiences. We analyzed both these sources of behavioral variation in two closely related brown frogs, Rana dalmatina and R. latastei. We raised tadpoles either with or without predators and tested them in open field trials both with and without predators. The effects of the raising environment were similar in the two species, whereas the effects of the testing arena differed. Both species decreased activity and increased speed and sinuosity with predators, but R. dalmatina moved always more and faster than R. latastei and it showed higher plasticity, larger variation among and within individuals, and relatively higher values in repeatability.

Amphibian larvae benefit from a warm environment under simultaneous threat from chytridiomycosis and ranavirosis

Rising temperatures can facilitate epizootic outbreaks, but disease outbreaks may be suppressed if temperatures increase beyond the optimum of the pathogens while still within the temperature range that allows for effective immune function in hosts. The two most devastating pathogens of wild amphibians, Batrachochytrium dendrobatidis (Bd) and ranaviruses (Rv), co‐occur in large areas, yet little is known about the consequences of their co‐infection and how these consequences depend on temperature. Here we tested how exposure to Bd and subsequent exposure to Rv, followed by treatment at elevated temperatures (28 and 30°C versus 22°C) affected Bd and Rv prevalence, infection intensities, and resulting mortalities in larval agile frogs and common toads. We found multiple pieces of evidence that the presence of one pathogen influenced the prevalence and/or infection intensity of the other pathogen in both species, depending on temperature and initial Rv concentration. Generally, the 30°C treatment lowered the prevalence and infection intensity of both pathogens and, in agile frogs, this was mirrored by higher survival. These results suggest that if temperatures naturally increase or are artificially elevated beyond what is ideal for both Bd and Rv, amphibians may be able to control infections and survive even the simultaneous presence of their most dangerous pathogenic enemies.

Embryonic exposure to native and alien predator cues tunes tadpole defensive behaviour

When exposed to predation risk, some amphibian species show innate responses, while others recognize their predators by learning. To explore the role played by each mechanism in the assessment of predation risk, we investigated the effects of embryonic and larval exposure to predator chemical cues on tadpole defensive responses, including behavioural, morphological and life history traits. In the first experiment, agile frog (Rana dalmatina) embryos were exposed to the odour of either native (Aeshna cyanea larvae) or alien (Procambarus clarkii) predators each day from egg collection to hatchling (14 days). Body measures (mass, developmental stage, body length, tail length and tail depth) were recorded at hatching and a behavioural test was conducted to explore tadpole responses to predator cues and the potential interaction with their previous embryonic experience. In general, embryonic conditioning did not affect life history traits, except for a slight reduction in tail depth:length ratio for tadpoles exposed to odonate odours. Controls (embryos treated with water) after hatchling reduced their activity when exposed to gammarid-fed odonate cues, suggesting that responses were at least partially innate. Tadpoles exposed to odonate cues as embryos showed a strong defensive response when exposed to dragonfly kairomones. Tadpoles exposed to gammarid-fed crayfish as embryos showed clear behavioural responses towards the same cue (irrespectively of predator diet). Overall, our results suggest that embryonic exposure may tune the defensive responses of the larval stage and early exposure to naïve stimuli may promote their cautionary associations with predation risk.

Efficacy of permanent wildlife fences as barriers to amphibian movement

To mitigate habitat fragmentation and roadkill, roads are increasingly equipped with wildlife fences and underpasses. However, the effectiveness of such fences in preventing road access for amphibians has not been tested under controlled conditions. In 2019 and 2020, we tested the efficacy of full panel fences of differing material, height, and shape (presence/absence of an overhang), to prevent road access for adult and juvenile amphibians. We selected five species according to locomotion mode: Natterjack toads (runners), European green toads (short-distance jumpers), agile frogs (proficient jumpers), American tree frogs (proficient climbers) and smooth newts (climbers). We found that Natterjack and green toads were unable to cross a concrete fence with a height of 13 and 24 cm, respectively. Addition of a 10 cm overhang reduced the height required to prevent crossing further to 10 and 17 cm, respectively. The ability of these less agile species to cross a certain fence height depended on body length. By contrast, jumping agile frogs and climbing tree frogs were not stopped by the greatest fence height tested (40 cm). However, addition of the overhang stopped the climbing tree frogs at a concrete fence height of 35 cm. An alternative metal fence (with overhang) was tested with some species and performed similar to the concrete fence (with overhang). Finally, the greatest concrete fence height passed by climbing juveniles was 20 cm (smooth newts). Hence, to stop amphibians from road crossing, we recommend the construction of durable (concrete or galvanized metal) and well-maintained fences with a minimum height of 40 cm with a 10 cm overhang.