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The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs ( Rana temporaria ) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species' activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

Chemical pollution, land cover change, and climate change have all been established as important drivers of amphibian reproductive success and phenology. However, little is known about the relative impacts of these anthropogenic stressors, nor how they may interact to alter amphibian population dynamics. Addressing this gap in our knowledge is important, as it allows us to identify and prioritise the most needed conservation actions. Here, we use long-term datasets to investigate landscape-scale drivers of variation in the reproductive success and phenology of UK Common frog (Rana temporaria) populations. Consistent with predictions, we found that increasing mean temperatures resulted in earlier initialisation of spawning, and earlier hatching, but these relationships were not consistent across all sites. Lower temperatures were also linked to increased spawn mortality. However, temperature increases were also strongly correlated with increases in urban area, arable area, and nitrate levels in the vicinity of spawning grounds. As with spawning and hatching, there was marked spatial variation in spawn mortality trends, where some sites exhibited steady increases over time in the proportion of dead or diseased spawn. These findings support previous work linking warming temperatures to shifts in timing of amphibian breeding, but also highlight the importance of assessing the effect of land use change and pollution on wild amphibian populations. These results have implications for our understanding of the response of wild amphibian populations to climate change, and the management of human-dominated landscapes for declining wildlife populations. [ABSTRACT FROM AUTHOR]

Chemical pollution, land cover change, and climate change have all been established as important drivers of amphibian reproductive success and phenology. However, little is known about the relative impacts of these anthropogenic stressors, nor how they may interact to alter amphibian population dynamics. Addressing this gap in our knowledge is important, as it allows us to identify and prioritise the most needed conservation actions. Here, we use long-term datasets to investigate landscape-scale drivers of variation in the reproductive success and phenology of UK Common frog (Rana temporaria) populations. Consistent with predictions, we found that increasing mean temperatures resulted in earlier initialisation of spawning, and earlier hatching, but these relationships were not consistent across all sites. Lower temperatures were also linked to increased spawn mortality. However, temperature increases were also strongly correlated with increases in urban area, arable area, and nitrate levels in the vicinity of spawning grounds. As with spawning and hatching, there was marked spatial variation in spawn mortality trends, where some sites exhibited steady increases over time in the proportion of dead or diseased spawn. These findings support previous work linking warming temperatures to shifts in timing of amphibian breeding, but also highlight the importance of assessing the effect of land use change and pollution on wild amphibian populations. These results have implications for our understanding of the response of wild amphibian populations to climate change, and the management of human-dominated landscapes for declining wildlife populations.

Ranaviruses have been involved in amphibian mass mortality events worldwide. Effective screening to control this pathogen is essential; however, current sampling methods are unsuitable for the detection of subclinical infections. Non-lethal screening is needed to prevent both further spread of ranavirus and losses of at-risk species. To assess non-lethal sampling methods, we conducted two experiments: bath exposing common frogs to RUK13 ranavirus at three concentrations, and exposing common toads to RUK13 or PDE18. Non-lethal sampling included buccal, digit, body and tank swabs, along with toe clips and stool taken across three time-points post-exposure. The presence/load of ranavirus was examined using quantitative PCR in 11 different tissues obtained from the same euthanised animals (incl. liver, gastro-intestinal tract and kidney). Buccal swab screening had the highest virus detection rate in both species (62% frogs; 71% toads) and produced consistently high virus levels compared to other non-lethal assays. The buccal swab was effective across multiple stages of infection and differing infection intensities, though low levels of infection were more difficult to detect. Buccal swab assays competed with, and even outperformed, lethal sampling in frogs and toads, respectively. Successful virus detection in the absence of clinical signs was observed (33% frogs; 50% toads); we found no difference in detectability for RUK13 and PDE18. Our results suggest that buccal swabbing could replace lethal sampling for screening and be introduced as standard practice for ranavirus surveillance. [ABSTRACT FROM AUTHOR]

The global trend of increasing environmental temperatures is often predicted to result in more severe disease epidemics. However, unambiguous evidence that temperature is a driver of epidemics is largely lacking, because it is demanding to demonstrate its role among the complex interactions between hosts, pathogens, and their shared environment. Here, we apply a three‐pronged approach to understand the effects of temperature on ranavirus epidemics in UK common frogs, combining in vitro, in vivo, and field studies. Each approach suggests that higher temperatures drive increasing severity of epidemics. In wild populations, ranavirosis incidents were more frequent and more severe at higher temperatures, and their frequency increased through a period of historic warming in the 1990s. Laboratory experiments using cell culture and whole animal models showed that higher temperature increased ranavirus propagation, disease incidence, and mortality rate. These results, combined with climate projections, predict severe ranavirosis outbreaks will occur over wider areas and an extended season, possibly affecting larval recruitment. Since ranaviruses affect a variety of ectothermic hosts (amphibians, reptiles, and fish), wider ecological damage could occur. Our three complementary lines of evidence present a clear case for direct environmental modulation of these epidemics and suggest management options to protect species from disease. [ABSTRACT FROM AUTHOR]

Amphibian populations have been declining globally over the past decades. The intensification of agriculture, habitat loss, fragmentation of populations and toxic substances in the environment are considered as driving factors for this decline. Today, about 50% of the area of Germany is used for agriculture and is inhabited by a diverse variety of 20 amphibian species. Of these, 19 are exhibiting declining populations. Due to the protection status of native amphibian species, it is important to evaluate the effect of land use and associated stressors (such as road mortality and pesticide toxicity) on the genetic population structure of amphibians in agricultural landscapes. We investigated the effects of viniculture on the genetic differentiation of European common frog (Rana temporaria) populations in Southern Palatinate (Germany). We analyzed microsatellite data of ten loci from ten breeding pond populations located within viniculture landscape and in the adjacent forest block and compared these results with a previously developed landscape permeability model. We tested for significant correlation of genetic population differentiation and landscape elements, including land use as well as roads and their associated traffic intensity, to explain the genetic structure in the study area. Genetic differentiation among forest populations was significantly lower (median pairwise FST = 0.0041 at 5.39 km to 0.0159 at 9.40 km distance) than between viniculture populations (median pairwise FST = 0.0215 at 2.34 km to 0.0987 at 2.39 km distance). Our analyses rejected isolation by distance based on roads and associated traffic intensity as the sole explanation of the genetic differentiation and suggest that the viniculture landscape has to be considered as a limiting barrier for R. temporaria migration, partially confirming the isolation of breeding ponds predicted by the landscape permeability model. Therefore, arable land may act as a sink habitat, inhibiting genetic exchange and causing genetic differentiation of pond populations in agricultural areas. In viniculture, pesticides could be a driving factor for the observed genetic impoverishment, since pesticides are more frequently applied than any other management measure and can be highly toxic for terrestrial life stages of amphibians.

The common frog (Rana temporaria) has been the focus of several broadscale phylogeographic studies, revealing a deep split between eastern and western European populations, most likely induced by the onset of the Pleistocene glaciations. However, the identification of glacial refugia, as well as the understanding of recolonization processes and their genetic legacy remain far from complete. A recent survey on Italian populations revealed a previously unrecognized Pleistocene refuge in the Italian peninsula and suggested the hypothesis of multiple separated microrefugia ('refugia-within refugia' model), but fine-scale studies required to confirm this hypothesis are lacking. We examined the phylogeographic structure of 54 common frog populations (540 individuals) by means of COI (cytochrome oxidase I) mitochondrial gene, focusing on a south-eastern Alpine region (Trentino, Italy) with an intensive sampling design. Phylogeographical reconstruction indicated the presence of three different COI lineages, exhibiting different levels of genetic diversity, and a contact zone in the eastern part of the region. Our data supported the scenario of multiple sub-refugia, probably located in the southern slopes of the Alpine chain, where the species survived the ice ages in fragmented populations. The recorded patterns are explained by the biogeographic peculiarity of the study region. [ABSTRACT FROM AUTHOR]

Graph-based analysis is a promising approach for analyzing the functional and structural connectivity of landscapes. In human-shaped landscapes, species have become vulnerable to land degradation and connectivity loss between habitat patches. Movement across the landscape is a key process for species survival that needs to be further investigated for heterogeneous human-dominated landscapes. The common frog ( Rana temporaria) was used as a case study to explore and provide a graph connectivity analysis framework that integrates habitat suitability and dispersal responses to landscape permeability. The main habitat patches influencing habitat availability and connectivity were highlighted by using the software Conefor Sensinode 2.2. One of the main advantages of the presented graph-theoretical approach is its ability to provide a large choice of variables to be used based on the study's assumptions and knowledge about target species. Based on dispersal simulation modelling in potential suitable habitat corridors, three distinct patterns of nodes connections of differing importance were revealed. These patterns are locally influenced by anthropogenic barriers, landscape permeability, and habitat suitability. And they are affected by different suitability and availability gradients to maximize the best possible settlement by the common frog within a terrestrial habitat continuum. The study determined the key role of landscape-based approaches for identifying the 'availability-suitability-connectivity' patterns from a local to regional approach to provide an operational tool for landscape planning. [ABSTRACT FROM AUTHOR]

Male mating success is generally governed by either female choice or male–male competition and multiple cues may influence both processes. Anuran amphibians are best known for acoustic communication but visual cues have also been found to play a role. Little is known, however, about visual cues in explosively breeding anurans in which sexual selection is based almost exclusively on male–male competition. During their search for mates, males may not distinguish between the sexes or species and mismating attempts are known to occur. Assuming that mismatings are costly for males, one would expect selection to favour a trait that facilitates sex recognition. We determined the colour of the explosively breeding common frog, Rana temporaria, during reproduction by spectrophotometry. While reflectance of all body parts in both sexes decreased from immigration to spawning, reflectance of male throats increased significantly. The mean luminance of male throats was more than twice that of female throats. Male throats contrasted most against male flanks with luminance contrast being significantly higher than chromatic contrast. In an experiment with individually marked frogs, throat luminance was not correlated with male size, body condition or the physical presence of females, whereas temperature showed a significantly positive effect. Our results give no hint that throats indicate male status or indicate male quality. Considering the high densities during breeding, our results suggest that bright male throats can act as a visual signal among males to facilitate gender recognition and avoid attacks by other males. [Copyright &y& Elsevier]

The inhibitor of gap junctions 18α-glycerretinic acid inhibits the maturation of follicle-enclosed common frog oocytes stimulated by low progesterone concentrations. The inhibitory effect of 18α-glycyrrhetinic acid does not depend on concentrations within the limits of 5–40 μM. The progesterone concentrations, at which oocyte maturation can be inhibited, differed markedly in different females. The inhibitory effects of actinomycin D (5 μg/ml) and 18α-glycyrrhetinic acid (5 μg/ml) were expressed when the same progesterone concentrations were used. When injected in an intact oocyte, Lucifer yellow was transferred into the follicle cells, thus suggesting the presence of gap junction between these latter and the oocyte. The data obtained suggest that the previously described transcription-dependent factor formed in the follicle cells under the influence of low progesterone concentrations and stimulating oocyte maturation is transferred in the oocyte via gap junctions. [ABSTRACT FROM AUTHOR]

Constructed wetlands are used as an effective measure to improve water quality and reduce nutrient runoff from agriculture. However, their role as alternative breeding sites for different amphibian species is rarely explored. We studied amphibians' colonization and breeding success in two in-stream free surface water wetlands in intensively managed agricultural landscape in south-eastern Estonia. Additionally, the sedimentation pond and a nearby farm pond were observed. The observations started a year after the establishment of the constructed wetlands in 2015 and lasted for four years. In Vända constructed wetlands, breeding of five amphibian species were observed, all of which were common to the area: smooth newt (Lissotriton vulgaris L.), common toad (Bufo bufo L.), common frog (Rana temporaria L.), moor frog (Rana arvalis Nilsson) and pool frog (Pelophylax lessonae Camerano). Threatened species, such as the common spadefoot toad (Pelobates fuscus Wagler) or the northern crested newt (Triturus cristatus Laur.), also present in this region, were not found in the study sites. Unlike the sedimentation and farm ponds, constructed wetlands with their shallow rapidly warming water and absence of fish, provided breeding habitat for amphibians, especially for common frog and moor frog. In addition, chemical analyses of the water showed that nutrient concentration in the surface water was relatively low, which should not impair the reproduction of amphibians, at least in the short-term. [ABSTRACT FROM AUTHOR]