Bufo Viridis Research Articles

An Easily Customizable Approach for Automated Species-Specific Detection of Anuran Calls Using the European Green Toad as an Example

Machine learning approaches for pattern recognition are increasingly popular. However, the underlying algorithms are often not open source, may require substantial data for model training, and are not geared toward specific tasks. We used open-source software to build a green toad breeding call detection algorithm that will aid in field data analysis. We provide instructions on how to reproduce our approach for other animal sounds and research questions. Our approach using 34 green toad call sequences and 166 audio files without green toad sounds had an accuracy of 0.99 when split into training (70%) and testing (30%) datasets. The final algorithm was applied to amphibian sounds newly collected by citizen scientists. Our function used three categories: “Green toad(s) detected”, “No green toad(s) detected”, and “Double check”. Ninety percent of files containing green toad calls were classified as “Green toad(s) detected”, and the remaining 10% as “Double check”. Eighty-nine percent of files not containing green toad calls were classified as “No green toad(s) detected”, and the remaining 11% as “Double check”. Hence, none of the files were classified in the wrong category. We conclude that it is feasible for researchers to build their own efficient pattern recognition algorithm.

A Pleistocene Amphibian Assemblage of the Taurida Cave, Crimea

Six amphibian taxa are described within the Pleistocene vertebrate assemblage of the Taurida cave (Crimean Peninsula): Lissotriton sp., Strauchbufo raddei, Bufotes viridis, Rana cf. macrocnemis, Pelophylax ridibundus, and Pelophylax sp. The possible presence of second species of the genus Rana is suggested. Three of described species do not occur on the Crimean Peninsula currently, but apparently inhabited it during the Early Pleistocene. The ranid frogs in Crimea were taxonomically more divers in the Pleistocene than today. Strauchbufo raddei and Bufotes viridis come from different periods of sediment accumulation of the Taurida cave, during the Early Pleistocene and the late Middle Pleistocene, respectively. The presence of the Mongolian toad, Strauchbufo raddei, not previously reported from Crimea, confirms a wide distribution of this species during the Early Pleistocene of Eastern Europe. The Taurida cave represents the first Pleistocene amphibian assemblage of Crimea.

Materials for the helminthofauna of the green toad Bufotes viridis (Anura, Amphibian) in the Republic of Dagestan

Materials for the helminthofauna of the green toad Bufotes viridis (Anura, Amphibian) in the Republic of Dagestan

A candidate sex determination locus in amphibians which evolved by structural variation between X- and Y-chromosomes

Most vertebrates develop distinct females and males, where sex is determined by repeatedly evolved environmental or genetic triggers. Undifferentiated sex chromosomes and large genomes have caused major knowledge gaps in amphibians. Only a single master sex-determining gene, the dmrt1-paralogue (dm-w) of female-heterogametic clawed frogs (Xenopus; ZW♀/ZZ♂), is known across >8740 species of amphibians. In this study, by combining chromosome-scale female and male genomes of a non-model amphibian, the European green toad, Bufo(tes) viridis, with ddRAD- and whole genome pool-sequencing, we reveal a candidate master locus, governing a male-heterogametic system (XX♀/XY♂). Targeted sequencing across multiple taxa uncovered structural X/Y-variation in the 5′-regulatory region of the gene bod1l, where a Y-specific non-coding RNA (ncRNA-Y), only expressed in males, suggests that this locus initiates sex-specific differentiation. Developmental transcriptomes and RNA in-situ hybridization show timely and spatially relevant sex-specific ncRNA-Y and bod1l-gene expression in primordial gonads. This coincided with differential H3K4me-methylation in pre-granulosa/pre-Sertoli cells, pointing to a specific mechanism of amphibian sex determination.

ВПЛИВ ПОГОДНИХ УМОВ НА ЧИСЕЛЬНІСТЬ АМФІБІЙ ТА РЕПТИЛІЙ В УМОВАХ ПРИРОДНОГО ЗАПОВІДНИКА «МЕДОБОРИ» ЗА ПЕРІОД 2016–2020 РОКІВ

Based on the results of long-term observations, the influence of weather conditions on the dynamics of the number and biodiversity of amphibians and reptiles over five years in the "Medobory" nature reserve was analyzed. The number, reproduction, metamorphosis stages, and distribution of amphibians and reptiles depend significantly on climatic conditions. Low temperatures in spring inhibit spawning and larval development. During critical periods, a low amount of precipitation (e.g., 15.7 mm in May 2016, 9.1 mm in April 2018, and 12.1 mm in April 2019, compared to the norm of 45 mm) also negatively affects amphibian populations. This factor led to the disappearance of Triturus cristatus Laur., Lissotriton vulgaris L. (an outbreak was noted in 2014), and Bufo viridis Laur. from the reserve (last observed at MG-1 in 2013), as well as the less common Hyla orientalis Bedriaga. Amphibians and reptiles were recorded during the active period along two regular herpetological routes, MG-1 and MG-2, and observations were made during field research, covering all areas suitable for the reproduction and habitat of amphibians. Eleven species of amphibians have been confirmed in the reserve, belonging to six families: Salamandridae, Bombinatoridae, Pelobatidae, Bufonidae, Hylidae, and Ranidae. Among these amphibians, five species are listed in Appendix II of the Berne Convention, and one species (T. cristatus Laur.) is included in the Red Book of Ukraine. The most common amphibian species are Rana temporaria L., Rana arvalis Nilsson, and Bufo bufo L. Frogs such as Pelophylax lessonae Camerano and Pelophylax ridibundus Pallas occasionally occur within the reserve, as they require an aquatic environment even after reproduction. There are seven species of reptiles in the reserve and one in the immediate vicinity, out of the ten recorded in the Ternopil region. These reptiles occupy the most suitable habitats for their survival. The species represent five families: Emydidae (freshwater turtles), Anguidae (spindly lizards), Lacertidae (true lizards), Colubridae (snakes), and Viperidae (vipers). Common species include Lacerta agilis Andrz., Natrix natrix L., and Anguis fragilis L. Six species of reptiles are included in Appendix II of the Berne Convention, and the common copperhead (Coronella austriaca Laur.) is included in the Red Book of Ukraine.

Metal nanoparticle-induced effects on green toads (Amphibia, Anura) under climate change: conservation implications.

The toxicity of aluminum oxide (Al2O3), copper oxide (CuO), iron oxide (Fe3O4), nickel oxide (NiO), zinc oxide (ZnO), and titanium dioxide (TiO2) nanoparticles (NPs) on amphibians and their interaction with high temperatures, remain unknown. In this study, we investigated the survival, developmental, behavioral, and histological reactions of Bufotes viridis embryos and larvae exposed to different NPs for a duration of 10 days, using lethal concentrations (LC25%, LC50%, and LC75% mg/L) under both ambient (AT: 18 °C) and high (HT: 21 °C) temperatures. Based on LC, NiONPs > ZnONPs > CuONPs > Al2O3NPs > TiO2NPs > Fe3O4NPs showed the highest mortality at AT. A similar pattern was observed at HT, although mortality occurred at lower concentrations and Fe3O4NPs were more toxic than TiO2NPs. The results indicated that increasing concentrations of NPs significantly reduced hatching rates, except for TiO2NPs. Survival rates decreased, abnormality rates increased, and developmental processes slowed down, particularly for NiONPs and ZnONPs, under HT conditions. However, exposure to low concentrations of Fe3O4NPs for up to 7 days, CuONPs for up to 72 h, and NiO, ZnONPs, and TiO2NPs for up to 96 h did not have a negative impact on survival compared with the control group under AT. In behavioral tests with larvae, NPs generally induced hypoactivity at AT and hyperactivity at HT. Histological findings revealed liver and internal gill tissue lesions, and an increase in the number of melanomacrophage centers at HT. These results suggest that global warming may exacerbate the toxicity of metal oxide NPs to amphibians, emphasizing the need for further research and conservation efforts in this context.

Records on malformations in Bufo bufo, Bufotes viridis and Bombina bombina from the Czech Republic

Records on malformations in Bufo bufo, Bufotes viridis and Bombina bombina from the Czech Republic

Spatiotemporal dynamics in the roosting ecology of the green toad: implications for urban planning and nature conservation

Continuously growing urbanisation and the ongoing rebuilding of urban habitats affect amphibians, the most threatened vertebrate groups. Even though the effects of urbanisation are mostly negative, some artificial components of the urban landscape may also display positive relationships with amphibian populations and therefore work in favour of their persistence in cities. Roosts provide protection from predation, desiccation and offer invertebrate prey availability; therefore, roost availability is an essential necessity of amphibian maintenance in urban areas. On the example of the European green toad Bufotes viridis, a typical urban dweller, we investigated roost-type occupancy and their distribution relative to a breeding pond during the active season (from June to October). The analysis was done with regard to the different life stages of individuals from three urban populations. Green toads used fissures situated in various man-made constructions (e.g., buildings, roads, pathways) for roosting cavities, however without any predominant pattern among toad life stages. The local architecture of the study sites mainly determined the type of occupied roosts. In adults and juveniles we recorded a gradually increased roost distance from breeding pond, while subadults were found roosting further from the water during the active season. Management measures aimed at maintaining a favourable state of green toad populations in urban environments should incorporate a complex of actions that consider general as well as the local ecological patterns of particular populations.

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.

Preliminary data on the diet composition and nesting behaviour of the Long‑legged Buzzard (Buteo rufinus) in south‑western Romania

The study of a nest of Long‑legged Buzzard Buteo rufinus was carried out using a trail camera and is one piece of a completely unknown jigsaw puzzle of diet and nesting behaviour in Romania. With a hatching success of 75%, we identified 3 hatched chicks from the 4 eggs. During the 10 days of monitoring, 34 preys brought to the nest were identified, with a peak of activity between 11:00–17:00 and a preference for the European Ground Squirrel Spermophilus citellus (61.76%). This was followed by the Domestic Pigeon Columba livia f. domestica and the Common Wood Pigeon C. palumbus (5.88%), and amphibians were represented by the European Green Toad Bufotes viridis (2.94%). There were 10 unidentified prey species (29.41%). We identified the presence of plastic and textile fibres in the nest and the risk of chick suffocation. We also improved our understanding of interspecific interactions and our observations highlighted the mutualism between the three species in the nest: Spanish Sparrow, House Sparrow and LLB. The removal of solitary trees from the agricultural landscape is a major threat in south‑western Romania, affecting the breeding population of the LLB, which, as in our case, nests on this type of support.

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).

Impact of Temperature on Larval Development of the Green Toad (Bufotes Viridis, Amphibia, Anura, Bufonidae) in Captivity

Identification of optimal temperature regimes to obtain the best values of productive indicators (reduction of development duration and feed costs, increase in size and survival rate) is the most crucial task for the development of amphibian breeding technologies. The paper presents the results of a study of the effect of temperature on the larval development of the green toad (Bufotes viridis). The offspring of a pair of green toads caught in the Kaluga region (Russia) served as the material for the work. Spawning was stimulated by injections of surfagon. During the transition of larvae to exogenous feeding, 144 individuals were selected from them for further growing. For experiments, containers measuring 39 × 28 × 28 cm filled with 18 liters of water were used. The animals were grown at a density of one larva per liter of water at the following temperatures: 20°C, 23°C, 26°C and 29°C. The larvae were fed daily with “TetraMin Flakes”. When the forelimbs appeared and when emerging from the water, the body length was measured in young toads. It is noted that with all temperature regimes (from 23°C to 29°C), the green toad larvae grew and metamorphosed successfully. Increasing temperature reduces the duration of larval development, but does not affect the size of the young emerging from the water. However, decreases in temperature below 23°C and increases above 26°C contribute to a decrease in survival. Based on the aforesaid, it is recommended that green toad larvae should be grown at 23–26°C to produce juveniles with body lengths of 11.20–20.83 mm and survival rates of 94–100% in 28–59 days.

Urban environment determines population genetics in the green toad, Bufotes viridis

Heavily urbanized areas can hinder dispersal and gene flow between amphibian populations. Given the growth potential of urbanization, it is important to examine how this specific environment shapes their genetic patterns at the local scale. The ability of the European green toad to successfully colonize large human settlements has been convincingly confirmed in the recent past, but little is known about its population genetics under these new conditions. In this study, we examined the effects of the environment on genetic variation, population structure, and the level of gene flow in populations of this amphibian in the city of Košice and the adjacent rural area (eastern Slovakia) using a set of neutral genetic markers. We found that urban populations had lower genetic variability than populations in adjacent rural areas; however, the degree of inbreeding was relatively low in all samples. Genetic differentiation was higher, and gene flow was more restricted in urban area, although geographic distances between sites were significantly less than in rural area (2–4 km versus 6–13 km). Our analyses suggested genetic isolation of urban populations at sites with less suitable habitat for green toads. In contrast, admixture of the population inhabiting the large city park, established on a former floodplain, with all rural populations was likely the result of an intense historical gene flow. The densely developed environment of the other urban sites likely presents a strong barrier to gene flow. The lack of suitable wetland habitat prior to development suggests that these sites were only recently colonized by a limited number of founders. Thus, we found differential effects of the city on the population structure of the green toad. Understanding current local genetic variation and structure is important for future conservation plans in urban environments.

Oswaldocruzia ukrainae (Nematoda: Molineidae)-A Parasite of European Green Toad Bufotes viridis: Morphological and Molecular Data.

Nematodes of the genus Oswaldocruzia are common parasites of the small intestine of amphibians and reptiles. Our recent molecular analysis of Oswaldocruzia nematodes revealed that only Oswaldocruzia filiformis, which possesses high morphological variability, parasitizes amphibians and reptiles in European Russia. Here we present the study of Oswaldocruzia nematodes from the European green toad Bufotes viridis (Anura, Bufonidae) collected at different localities of the Middle Volga region in 2018-2022. We analyzed the morphological characteristics of the Oswaldocruzia spp. taxonomy together with novel molecular phylogenetic data. The data on phylogenetic analysis (based on partial CoxI mtDNA gene sequences) showed that Bufotes viridis is parasitized by two Oswaldocruzia species, the host-specific parasite Oswaldocruzia ukrainae and species generalist Oswaldocruzia filiformis. Broad morphological variability was revealed in O. ukrainae nematodes both from the same host specimen and from various toad individuals from different localities. Our results highlight the need for further biodiversity research of morphologically similar Oswaldocruzia species from amphibians and reptiles in the Western Palearctic using molecular genetic methods.

Causes for the High Mortality of European Green Toad Tadpoles in Road Stormwater Ponds: Pollution or Arrival of a New Predator?

Declining wetland areas cause many amphibian species to breed inside stormwater ponds (SWPs), which have been constructed alongside major roads to collect and retain polluted road runoff water. However, the suitability of such artificial ponds as a breeding habitat for amphibians remains unclear. Recently, a study found a very low survival rate of European green toad tadpoles (Bufotes viridis) inside SWPs, presumably because of high sediment pollution and/or the presence of a leech Helobdella stagnalis. To establish the effects of sediment pollution and leech presence on tadpole growth and survival, we exposed 480 green toad tadpoles to a number of controlled conditions inside holding tanks. We tested the following conditions: (1) ‘control’ (clean sediment + clean water); (2) ‘leech’ (clean sediment + clean water + leeches); (3) ‘SWP sediment’ (clean water + polluted sediment); and (4) ‘SWP sediment + leech’ (clean water + polluted sediment + leeches). Tadpole size and survival was monitored until metamorphosis and, individuals participated in swim tests and respirometry trials to the test potential effects of pollution on their escape capacity and metabolic rate. We found that the growth rate of tadpoles exposed to the SWP sediment (condition 3) was increased, while pollution had no effect on survival. By contrast, leeches heavily preyed upon tadpoles, leaving no survivors in conditions 2 and 4. Tadpoles swim speed and metabolic rate of toadlets did not differ between the ‘control’ and ‘SWP sediment’ group, the only conditions with surviving individuals. Our study found that leeches had the strongest effect on tadpole survival and were likely responsible for the low survival rates in SWPs observed recently. Hence, we suggest that adequate management measures are needed to limit leech penetration inside SWPs (frequent dredging/draining) to prevent these artificial structures from becoming an ecological trap for locally endangered amphibians but rather a base to help in their recovery.

Spatiotemporal Patterns and Road Mortality Hotspots of Herpetofauna on a Mediterranean Island

Traffic intensification, often in conjunction with habitat fragmentation, has caused frequent roadkill incidents, particularly among reptiles, amphibians, and other taxa. Herpetofauna species, which are slow moving and habitat dependent, are particularly susceptible to these incidents because they often use roads during thermoregulation. Lesvos, the eighth-largest Mediterranean island, boasts a biodiversity that surpasses most other Mediterranean islands of similar or larger size, with a plethora of herpetofauna species inhabiting its terrain. In recent years, new roads were constructed on Lesvos, which are considered to be one of the most important factors that negatively affect the island’s wildlife as they increase the non-natural mortality of animals, are obstacles to their mobility, and reduce the connectivity of populations by limiting their dynamics. In the present study, we examined the road mortality of amphibians and reptiles by analyzing (a) the temporal and seasonal patterns of such incidents, (b) the relationship between roadside habitats and road network characteristics with the roadkilled herpetofauna species, and (c) their spatial distribution on Lesvos during the years 2009–2012 and 2016–2019. To identify significant clusters of reptile and amphibian roadkills, we performed hotspot analysis by utilizing kernel density estimation and Getis Ord Gi* statistics. We recorded a total of 330 roadkills among 20 species, with the highest rates emerging among the European green toad (Bufotes viridis), the European glass lizard (Pseudopus apodus), the Rhodos green lizard (Lacerta diplochondrodes), and the snake-eyed lizard (Ophisops elegans). Spatial statistical analysis revealed that roads close to herpetofauna habitats exhibit statistically significant clusters of roadkills that intensify during the spring season. Regular monitoring and mapping of herpetofauna road mortality will enable the implementation of management strategies to mitigate the negative impact of this phenomenon.

Temporal and spatial variations in local sex ratios in a suburban population of the European green toad Bufotes viridis

BackgroundSex ratios of animal populations are important factors of population demographics. In pond-breeding amphibians, the operational sex ratio (OSR) among the breeding population is usually male-biased. Also, in European green toads (Bufotes viridis), males usually outnumber females at breeding sites, while the sex ratio of the total adult population (ASR) is assumed to be balanced. It has been suggested that sex-specific breeding behavior causes male-predominance at the breeding sites. We used a dataset of 5 years of street patrols to test this hypothesis. For this we analyzed local sex ratios of green toads in terrestrial habitats and at two artificial breeding ponds. We expected temporal and/or spatial changes of local sex ratios which would indicate sex dependent differences in breeding behavior.ResultsOverall observed ASR among 2111 green toads, counted in the course of street patrols from 2016 to 2020, was slightly male-biased (ASR = 0.56, annual ASRs = 0.49–0.63). Based on the data of 1631 toads (920 males, 711 females) captured within a radius of 300 m around nine main breeding sites, temporal and spatial variations in local ASRs were evaluated. Resulting values were compared to the calculated OSR at two artificial breeding ponds in 2021 (645 adult: 553 males, 92 females). Estimates predict more equally distributed females and males prior to the main breeding season. During breeding season, males predominated at both breeding sites (B1: 0.83, B2: 0.89), whereas females are estimated to outnumber males in terrestrial habitats. Proportions of females highly significantly increased with advancing time of the year and increasing distance to the breeding sites. While males tended to accumulate in proximity to water bodies, females dispersed soon after breeding to more distant areas.ConclusionsObserved sex ratios in the studied green toad population changed with time and sampling site, deviating from the population-wide sex ratio. Expanding sampling effort in amphibian conservation assessments in time and space, i.e., outside the main breeding season and away from the breeding sites, would be important to encompass such variations.

Telemetry and Accelerometer Tracking of Green Toads in an Urban Habitat: Methodological Notes and Preliminary Findings.

Advancements in tracking technologies provide an increasingly important tool in animal monitoring and conservation that can describe animal spatial behavior in native habitats and uncover migratory routes that otherwise may be difficult or impossible to map. In addition, high-resolution accelerometer sensors provide powerful insights into animal activity patterns and can help to identify specific behaviors from accelerometer profiles alone. Previously, such accelerometers were restricted to larger animals due to size and mass constraints. However, recent advances make it possible to use such devices on smaller animals such as the European green toad (Bufotes viridis), the focus of our current study. We deploy custom made tracking devices, that consist of very-high-frequency transmitters and tri-axial accelerometers, to track toads in their native urban environment in Vienna (Austria). A total of nine toads were tracked, ranging from three to nine tracking days per individual during the post-breeding season period. We demonstrate that our devices could reliably monitor toad movement and activity during the observation period. Hence, we confirmed the predominantly nocturnal activity patterns and recorded low overall movement at this urban site. Accelerometer data revealed that toads exhibited brief but intense activity bursts between 10 pm and midnight, resting periods during the night and intermittent activity during the day. Positional tracking alone would have missed the major activity events as they rarely resulted in large positional displacements. This underscores the importance of and value in integrating multiple tracking sensors for studies of movement ecology. Our approach could be adapted for other amphibians or other animals with mass constraints and may become standard monitoring equipment in the near future.

Bufadienolides from the Bufo viridis toad venom exert cytotoxic effects on cancer cells by inducing cell apoptosis and cell cycle arrest

A series of bufadienolides were isolated from the Bufo viridis toad venom, and their cytotoxic activities against three human cancer cell lines (HeLa, HT-29, MCF7) and a non-cancer cell line (L-O2) were explored using the MTT assay in vitro. All of nine compounds exhibited cytotoxic activities against the three cancer cell lines, with compound D4 exhibiting potent cytotoxic activity against HeLa cells and was better than positive control. Herein, we further evaluated the effect of compound D4 on HeLa cells. The results revealed that compound D4 has excellent cytotoxic effect on HeLa cells by inhibiting cell colony formation and migration, promoting cell apoptosis, increasing reactive oxygen species (ROS) levels and arresting of HeLa cells in S and G2/M phases. These findings encourage further work on the chemistry and bioactivity of the Bufo viridis toad venom.

Land-use preferences of the European green toad (Bufotes viridis) in the city of Vienna (Austria): the importance of open land in urban environments

Urban areas are increasing worldwide, which poses threats to animal wildlife. However, in certain cases cities can provide refuges for endangered animals. The European green toad (Bufotes viridis) is one of such examples, which is known from cities throughout their distribution. In contrast, considerable areas of their former (primary) habitats have been degraded. The primary habitats of this species include steppes and wild river floodplains, both characterized by dynamic changes and the presence of open areas. We used available green toad observation data (2007–2020) to model the effects of land-use types on occurrence probability in the city of Vienna. Forest and densely populated areas were highly significantly negatively associated with green toad presence, while transformation/construction site areas showed a strong positive effect. Such occurrence pattern might be characteristic for early succession species, which depend on stochastic environmental disturbances (e.g., droughts and floods) in their primary habitats. We argue that urban landscape planning should appreciate the potential ecological value of open land in cities which is either in a transition phase or a permanent ‘wasteland’. Ecological managing of such landscape could vastly increase urban biodiversity.