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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 co-infection and elevated temperatures (28 and 30°C vs. 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.

Emerging infectious diseases are major drivers of global and local amphibian biodiversity loss. Therefore, developing effective disinfection methods to manage the impact of diseases in wild and captive "ark" populations are an important goal in amphibian conservation. While chemical disinfectants have been used safely and effectively in larval and adult amphibians infected with pathogenic microbes, their applicability to amphibian egg masses has remained untested. To bridge this gap, we exposed embryos of the common toad (Bufo bufo) and agile frog (Rana dalmatina) experimentally to three widely used disinfectants: voriconazole, chloramphenicol and chlorogen-sesquihydrate. For 3 days we exposed portions of egg masses to these disinfectants at 1×, 2×, 5× and 10× the concentration recommended for the disinfection of tadpoles and adults. Subsequently, we recorded embryonic and larval survival, as well as larval body mass and the incidence of abnormalities 12 days after hatching. Application of voriconazole had species- and concentration-dependent negative impacts on survival and body mass, and caused marked malformations in the viscerocranial structure of B. bufo tadpoles. Exposure to chlorogen-sesquihydrate also resulted in significant mortality in B. bufo embryos and negatively affected body mass of R. dalmatina larvae. Chloramphenicol had little negative effects on embryos or larvae in either species. Based on these results, the application of voriconazole and chlorogen-sesquihydrate cannot be recommended for the disinfection of amphibian eggs, whereas treatment with chloramphenicol appears to be a safe method for eliminating potential pathogens from anuran egg masses and their immediate aquatic environment.

Many organisms synthesize secondary metabolites against natural enemies. However, to which environmental factors the production of these metabolites is adjusted to is poorly investigated in animals, especially so in vertebrates. Bufadienolides are steroidal compounds that are present in a wide range of plants and animals and, if present in large quantities, can provide protection against natural enemies, such as pathogens. In a correlative study involving 16 natural populations we investigated how variation in bufadienolide content of larval common toads (Bufo bufo) is associated with the bacterial community structure of their aquatic environment. We also evaluated pond size, macrovegetation cover, and the abundance of predators, conspecifics and other larval amphibians. We measured toxin content of tadpoles using HPLC-MS and determined the number of bufadienolide compounds (NBC) and the total quantity of bufadienolides (TBQ). AICc-based model selection revealed strong relationships of NBC and TBQ with bacterial community structure of the aquatic habitat as well as with the presence of conspecific tadpoles. The observed relationships may have arisen due to adaptation to local bacterial communities, phenotypic plasticity, differential biotransformation of toxin compounds by different bacterial communities, or a combination of these processes. Bacterial groups that contribute to among-population variation in toxin content remain to be pinpointed, but our study suggesting that toxin production may be influenced by the bacterial community of the environment represents an important step towards understanding the ecological and evolutionary processes leading to microbiota-mediated variation in skin toxin profiles of aquatic vertebrates. Electronic supplementary material The online version of this article (10.1007/s10886-020-01184-4) contains supplementary material, which is available to authorized users.

Background Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, one of the major causes of worldwide amphibian biodiversity loss. Many amphibians exhibit skin-based chemical defences, which may play an important role against invading pathogens, but whether the synthesis of these chemical compounds is enhanced or suppressed in the presence of pathogens is largely unknown. Here we investigated direct and indirect effects of larval exposure to the globally distributed and highly virulent Bd-GPL strain on skin secreted chemical defences and life history traits during early ontogeny of agile frogs (Rana dalmatina) and common toads (Bufo bufo). Results Exposure to Bd during the larval stage did not result in enhanced synthesis of the antimicrobial peptide Brevinin-1 Da in R. dalmatina tadpoles or in increased production of bufadienolides in B. bufo tadpoles. However, exposure to Bd during the larval stage had a carry-over effect reaching beyond metamorphosis: both R. dalmatina and B. bufo froglets contained smaller quantities of defensive chemicals than their Bd-naïve conspecifics in the control treatment. Prevalence of Bd and infection intensities were very low in both larvae and metamorphs of R. dalmatina, while in B. bufo we observed high Bd prevalence and infection intensities, especially in metamorphs. At the same time, we did not find a significant effect of Bd-exposure on body mass or development rate in larvae or metamorphs in either species. Conclusions The lack of detrimental effect of Bd-exposure on life history traits, even parallel with high infection intensities in the case of B. bufo individuals, is surprising and suggests high tolerance of local populations of these two species against Bd. However, the lowered quantity of defensive chemicals may compromise antimicrobial and antipredatory defences of froglets, which may ultimately contribute to population declines also in the absence of conspicuous mass-mortality events.

High‐quality information on predator–prey relationships is fundamental in understanding food webs, community assembly and ecosystem functioning. Recent analytical advances have made it possible to develop new trait‐based approaches to study trophic relationships and evaluate trait matching between predators and prey. Here, we develop a novel analytical approach based on generalized linear mixed‐effects models (GLMM) to test the importance of prey availability and to identify the set of prey traits that best explain the occurrence and number of prey in the predator's diet. We demonstrate that the approach by using an extensive dataset on prey availability, prey traits and gut content collected in all known populations of Vipera graeca, a little‐known, endangered snake of alpine grasslands in the Pindos Mountains of the Balkan Peninsula. We show that V. graeca is a unique, venomous snake specialized on bush‐crickets and grasshoppers (Orthoptera). Prey selection GLMMs showed that the ideal prey of V. graeca is abundant, large‐bodied, has poor escape abilities (flightless, slow‐moving and bad jumper) and prefers loose grasslands (as opposed to bare ground/rock or closed sward). Vipers restrict their feeding to periods of high Orthoptera abundance in the late summer and need to reach a certain body size to become able to catch large‐sized prey. Our analytical approach provides a framework for trait matching between predators and prey and unprecedented fine‐scale information on the importance of prey traits in prey selection by a specialist predator. The narrow trophic niche of V. graeca likely increases the vulnerability of this cold‐adapted snake to extinction. A free Plain Language Summary can be found within the Supporting Information of this article.

Extreme temperatures during heat waves can induce mass-mortality events, but can also exert sublethal negative effects by compromising life-history traits and derailing sexual development. Ectothermic animals may, however, also benefit from increased temperatures via enhanced physiological performance and the suppression of cold-adapted pathogens. Therefore, it is crucial to address how the intensity and timing of naturally occurring or human-induced heat waves affect life-history traits and sexual development in amphibians, to predict future effects of climate change and to minimise risks arising from the application of elevated temperature in disease mitigation. We raised agile frog (Rana dalmatina) and common toad (Bufo bufo) tadpoles at 19 °C and exposed them to a simulated heat wave of 28 or 30 °C for six days during one of three ontogenetic periods (early, mid or late larval development). In agile frogs, exposure to 30 °C during early larval development increased mortality. Regardless of timing, all heat-treatments delayed metamorphosis, and exposure to 30 °C decreased body mass at metamorphosis. Furthermore, exposure to 30 °C during any period and to 28 °C late in development caused female-to-male sex reversal, skewing sex ratios strongly towards males. In common toads, high temperature only slightly decreased survival and did not influence phenotypic sex ratio, while it reduced metamorph mass and length of larval development. Juvenile body mass measured two months after metamorphosis was not adversely affected by temperature treatments in either species. Our results indicate that heat waves may have devastating effects on amphibian populations, and the severity of these negative consequences, and sensitivity can vary greatly between species and with the timing and intensity of heat. Finally, thermal treatments against cold-adapted pathogens have to be executed with caution, taking into account the thermo-sensitivity of the species and the life stage of animals to be treated.

Abstract Parasites, including viruses, bacteria, fungi, protists, helminths, and arthropods, are ubiquitous in the animal kingdom. Consequently, hosts are frequently infected with more than one parasite species simultaneously. The assessment of such co-infections is of fundamental importance for disease ecology, but relevant studies involving non-domesticated animals have remained scarce. Many amphibians are in decline, and they generally have a highly diverse parasitic fauna. Here we review the literature reporting on field surveys, veterinary case studies, and laboratory experiments on co-infections in amphibians, and we summarize what is known about within-host interactions among parasites, which environmental and intrinsic factors influence the outcomes of these interactions, and what effects co-infections have on hosts. The available literature is piecemeal, and patterns are highly diverse, so that identifying general trends that would fit most host–multiparasite systems in amphibians is difficult. Several examples of additive, antagonistic, neutral, and synergistic effects among different parasites are known, but whether members of some higher taxa usually outcompete and override the effects of others remains unclear. The arrival order of different parasites and the time lag between exposures appear in many cases to fundamentally shape competition and disease progression. The first parasite to arrive can gain a marked reproductive advantage or induce cross-reaction immunity, but by disrupting the skin and associated defences (i.e., skin secretions, skin microbiome) and by immunosuppression, it can also pave the way for subsequent infections. Although there are exceptions, detrimental effects to the host are generally aggravated with increasing numbers of co-infecting parasite species. Finally, because amphibians are ectothermic animals, temperature appears to be the most critical environmental factor that affects co-infections, partly via its influence on amphibian immune function, partly due to its direct effect on the survival and growth of parasites. Besides their importance for our understanding of ecological patterns and processes, detailed knowledge about co-infections is also crucial for the design and implementation of effective wildlife disease management, so that studies concentrating on the identified gaps in our understanding represent rewarding research avenues. Graphical Abstract

Analyzing hybrid zones between previously isolated lineages allows insight into processes determining the fate of re-encounter of these taxa. The distributions of Fire-bellied (Bombina bombina) and Yellow-bellied toads (B. variegata) meet in the Carpathian Basin resulting in a narrow contact zone in the foothill regions, where hybrids often appear. Our aim was to explore a transient zone between B. bombina and B. variegata within the Carpathian Basin along a transect including the Börzsöny Hills in Hungary and Krupinská Planina in Slovakia. We visited 28 locations in these areas and collected altogether 230 specimens, photographed and sampled them using buccal swabs. In order to distinguish between the two species and determine hybrid individuals, we used mitochondrial markers and designed a novel technique based on the restriction of the Ncx-1 gene. The geographical distribution pattern of these two species delivered unexpected results, as Börzsöny Hills was completely colonised by B. bombina including locations which can be classified as typical habitats of B. variegata. Moreover, in Krupinská Planina many locations were also colonised by B. bombina, including high altitude ponds. The most remote sites still harbour B. variegata populations, but seven of these were found with hybrid individuals. This pattern may indicate the northward and altitudinal range expansion of B. bombina and the colonisation of habitats of its sister species in these areas. Our results warrant enhanced attention to hybrid zones, where introgression and changes in population composition may reflect recent rapid environmental alterations and redirect conservation focus.

Anthropogenic environmental change poses a special threat to species in which genetic sex determination can be overwritten by the thermal and chemical environment. Endocrine disrupting chemicals as well as extreme temperatures can induce sex reversal in such species, with potentially wide-ranging consequences for fitness, demography, population viability and evolution. Despite accumulating evidence suggesting that chemical and thermal effects may interact in ecological contexts, little is known about their combined effects on sex reversal. Here we assessed the simultaneous effects of high temperature (female-to-male sex-reversing agent) and 17α-ethinylestradiol (EE2), a widespread xenoestrogen (male-to-female sex-reversing agent), on sexual development and fitness-related traits in agile frogs (Rana dalmatina). We exposed tadpoles to a six-days heat wave (30 °C) and/or an ecologically relevant concentration of EE2 (30 ng/L) in one of three consecutive larval periods, and diagnosed sex reversals two months after metamorphosis using species-specific markers for genetic sexing. We found that high temperature induced female-to-male sex reversal, decreased survival, delayed metamorphosis, decreased body mass at metamorphosis, and increased the proportion of animals that had no fat bodies, while EE2 had no effect on these traits. Simultaneous exposure to heat and EE2 had non-additive effects on juvenile body mass, which were dependent on treatment timing and further complicated by a negative effect of sex reversal on body mass. These results show that environmentally relevant exposure to EE2 does not diminish the female-to-male sex-reversing effects of high temperature. Instead, our findings on growth suggest that climate change and chemical pollution may have complex consequences for individual fitness and population persistence in species with environment-sensitive sex determination.

Many animals display spectacular adjustments of their phenotype to changing environmental conditions in terms of altered behaviour, physiology and morphology, but whether similar plasticity in chemical defences is taxonomically widespread as well has remained unknown. Bufadienolides, toxins produced by some anuran amphibians, are known to be effective against many predators, but they can also contribute to defences against microorganisms. We experimentally investigated if qualitative and/or quantitative changes in the chemical defences of Bufo bufo tadpoles are induced by differences in the microbiota of their environment. We reared animals in purified tap water, or in pond water containing lowered or natural quantities of the natural microbiota, and measured the number of compounds and total quantity of bufadienolides at three ontogenetic stages during larval development, as well as 10 days after metamorphosis. We observed a significant decrease in the number of compounds, and a parallel increase in the total quantity of bufadienolides after metamorphosis, independent from treatments. The quantity of individual bufadienolide compounds also changed during ontogeny independently from treatments. Our results, thus, suggest that although bufadienolide production changes during early ontogeny, it is not adjusted to the microbiota present in the environment.

Despite a steeply increasing number of ecotoxicological studies on the effects of pesticides on nontarget organisms, studies assessing the adequacy and reliability of different experimental approaches have remained scarce. We scrutinized effects of a glyphosate-based herbicide on larvae of two European anuran amphibians by estimating species-specific LC50 values, assessing how an additional stress factor may influence outcomes, and investigating whether replicate experiments yielded qualitatively the same results. We exposed Rana dalmatina and Bufo bufo tadpoles to two predator treatments (no predator vs. predator chemical cues) combined with varying herbicide concentrations, repeated the experiment with a subset of the experimental treatments and partly with slight modifications 1 week later and assessed survival. Our results indicated that the herbicide was moderately toxic to tadpoles. The presence of predator chemical cues did not affect the lethality of the herbicide in either species. The estimated sensitivity of R. dalmatina tadpoles varied considerably across experiments, whereas in case of B. bufo LC50 values remained very similar. Our results suggest that differences in the experimental setup may often have no influence on the measured effects of pesticides, whereas replicated experiments can deliver widely differing results in other cases, perhaps depending on the studied species, the population origin of the tested individuals, or the test conditions. This draws attention to the suggestion that strict standardization may not deliver widely applicable insights into the toxicity of contaminants and, instead, intentionally introducing variation into the design of ecotoxicological experiments and replicating entire experiments may prove highly beneficial.

The worldwide en masse application of pesticides and the frequently reported malign effects on several non-target organisms underpin the importance of ecotoxicological research on these anthropogenic pollutants. Previous studies showed that sensitivity to herbicides can vary widely depending on additional stress factors, on the species and even on the population investigated. However, there is little information about how sensitivity changes during ontogeny, and how the duration of exposure is linked to the magnitude of malign effects, even though this knowledge would be important for the interpretation of toxicity test results and for formulating recommendations regarding the timing of pesticide application. We exposed tadpoles of the common toad (Bufo bufo) to three concentrations (0, 2 and 4mg a.e./L) of a glyphosate-based herbicide during the 1st, 2nd, 3rd, 4th, or 5th period of larval development or during the entire experiment, and measured survival, time until metamorphosis and body mass at metamorphosis to estimate fitness-consequences. Younger tadpoles were more sensitive to the herbicide in all measured traits than older ones, and this age-dependence was especially pronounced at the high herbicide concentration. Furthermore, tadpoles exposed to the herbicide during the entire experiment developed slower than tadpoles exposed only early on, but we did not observe a similar effect either on body mass or survival. The observed age-dependence of sensitivity to herbicides draws attention to the fact that results of toxicity tests obtained for one age-class are not necessarily generalizable across ontogeny. Also, the age of test animals has to be considered when planning ecotoxicological studies and interpreting their results. Finally, taking into account the temporal breeding habits of local amphibians when planning pesticide application would be highly favourable: if tadpoles would not get exposed to the herbicide during their most sensitive early development, they would sustain less anthropogenic damage from our efforts of controlling weeds.

A keteltűeket veszelyeztető kitridiomikozis korokozoja, a Batrachochytrium dendrobatidis (Bd; Longcore, Pessier & D. K. Nichols, 1999) nevű gombafaj bizonyitottan jelen van a Karpat-medenceben is, ugyanakkor a szamlajara irhato drasztikus allomanycsokkenest itt ez idaig nem tapasztaltunk. Ennek egyik oka lehet a honos populaciok magas toleranciaja a betegseggel szemben. Kiserletunkben barna varangy es erdei beka ebihalait fertőztuk egy magas virulenciaju Bd torzzsel. A gomba egyik fajnal sem csokkentette a tulelest, a fejlődesi sebesseget es az atalakulas utani tomeget. Ugyanakkor a barna varangy egyedeknel az erdei bekaknal mert fertőzottseg sokszorosat figyeltuk meg. Ugy tűnik, hogy a hazai barna varangy populaciok magas toleranciaval, az erdei beka populaciok pedig magas rezisztenciaval rendelkeznek a betegseggel szemben.

Pesticides can exert negative effects on aquatic organisms at very low concentrations. While several prey taxa are frequently used as models in ecotoxicology studies, there is little information about the pesticide-sensitivity of predators. We examined the effects of a frequently applied glyphosate-based herbicide on two common aquatic predators: larval Aeshna cyanea dragonflies and adult male Lissotriton vulgaris newts, which are top predators in ephemeral water bodies lacking fishes. We exposed predators to the herbicide for 18 days under laboratory conditions and measured potential effects on survival, activity, body mass and predatory activity. To maximize detectability of effects, we applied the herbicide at a concentration of 6.5 mg a. e. glyphosate/L, corresponding to the highest concentration expected in nature. Our results showed that the tested herbicide formulation did not have severe effects on any of the measured fitness-related traits. Results of the present study support the hypothesis that the tested species are insensitive to the herbicide and are able to fulfil their important ecological role of top-down regulation even in highly contaminated habitats. However, potential long-term or indirect effects of the herbicide on the fitness of aquatic predators remain unknown.

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 6.5 mg 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.

It has been implied that the application of pesticides is involved in the world-wide decline of biodiversity, but little is known about the influence of these chemicals on key predators of temporary wetlands. The direct impacts were examined of a frequently applied glyphosate-based herbicide on larval Aeshna cyanea (Muller, 1764; Odonata, Insecta) and adult male Lissotriton vulgaris (Linnaeus, 1758; Caudata, Amphibia), 2 top predators of Central European ephemeral ponds. The effects of herbicide exposure were measured on survival, behavior, body mass change, and predatory activity in an outdoor mesocosm experiment lasting for 17 d. No significant effects of exposure were observed in either predator species. The results suggest that the herbicide has no immediate effect on the predators studied at environmentally relevant concentrations and that these predators can also fulfill their top-down regulatory role in contaminated ecosystems.

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.

The widespread application of pesticides emphasises the importance of understanding the impacts of these chemicals on natural communities. The most commonly applied broad-spectrum herbicides in the world are glyphosate-based herbicides, which have been suggested to induce significant behavioural changes in non-target organisms even at low environmental concentrations. To scrutinize the behavioural effects of herbicide-exposure we exposed agile frog (Rana dalmatina) tadpoles in an outdoor mesocosm experiment to three concentrations of a glyphosate-based herbicide (0, 2 and 6.5 mg acid equivalent (a.e.) / L). To assess whether anti-predator behaviour is affected by the pesticide, we combined all levels of herbicide-exposure with three predator treatments (no predator, caged Aeshna cyanea dragonfly larvae or Lissotriton vulgaris newt adults) in a full factorial design. We observed hiding, activity, proximity to the predator cage and vertical position of tadpoles. We found that at the higher herbicide concentration tadpoles decreased their activity and more tadpoles were hiding, and at least at the lower concentration their vertical position was closer to the water surface than in tadpoles of the control treatment. Tadpoles also decreased their activity in the presence of dragonfly larvae, but did not hide more in response to either predator, nor did tadpoles avoid predators spatially. Further, exposure to the herbicide did not significantly influence behavioural responses to predation threat. Our study documents a definite influence of glyphosate-based herbicides on the behaviour of agile frog tadpoles and indicates that some of these changes are similar to those induced by dangerous predators. This may suggest that the underlying physiological mechanisms or the adaptive value of behavioural changes may similar.

It has been implied that the application of pesticides is involved in the world-wide decline of biodiversity, but little is known about the influence of these chemicals on key predators of temporary wetlands. The direct impacts were examined of a frequently applied glyphosate-based herbicide on larval Aeshna cyanea (Müller, 1764; Odonata, Insecta) and adult male Lissotriton vulgaris (Linnaeus, 1758; Caudata, Amphibia), 2 top predators of Central European ephemeral ponds. The effects of herbicide exposure were measured on survival, behavior, body mass change, and predatory activity in an outdoor mesocosm experiment lasting for 17 d. No significant effects of exposure were observed in either predator species. The results suggest that the herbicide has no immediate effect on the predators studied at environmentally relevant concentrations and that these predators can also fulfill their top-down regulatory role in contaminated ecosystems.