Metabolites of Xenorhabdus bacteria are potent candidates for mitigating amphibian chytridiomycosis.

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has caused extreme losses in amphibian biodiversity. Finding bacteria that produce metabolites with antifungal properties may turn out to be invaluable in the fight against this devastating disease. The entomopathogenic bacteria, Xenorhabdus szentirmaii and X. budapestensis produce secondary metabolites that are effective against a wide range of fungal plant pathogens. To assess whether they may also be effective against Bd, we extracted cell-free culture media (CFCM) from liquid cultures of X. szentirmaii and X. budapestensis and tested their ability to inhibit Bd growth in vitro. As a second step, using juvenile common toads (Bufo bufo) experimentally infected with Bd we also tested the in vivo antifungal efficacy of X. szentirmaii CFCM diluted to 2 and 10% (v/v), while also assessing possible malign side effects on amphibians. Results of the in vitro experiment documented highly effective growth inhibition by CFCMs of both Xenorhabdus species. The in vivo experiment showed that treatment with CFCM of X. szentirmaii applied at a dilution of 10% resulted in infection intensities reduced by ca. 73% compared to controls and to juvenile toads treated with CFCM applied at a dilution of 2%. At the same time, we detected no negative side effects of treatment with CFCM on toad survival and development. Our results clearly support the idea that metabolites of X. szentirmaii, and perhaps of several other Xenorhabdus species as well, may prove highly useful for the treatment of Bd infected amphibians.

In vitro thermal tolerance of a hypervirulent lineage of Batrachochytrium dendrobatidis: Growth arrestment by elevated temperature and recovery following thermal treatment.

Chytridiomycosis, an emerging infectious disease caused by Batrachochytrium dendrobatidis (Bd), poses a serious threat to amphibians. The thermal optimum of Bd is lower than that of most amphibians, providing an opportunity to cure infected individuals with elevated temperature. However, this approach presupposes detailed knowledge about the thermal tolerance of the fungus. To determine the temperature that may effectively reduce infection burdens in vivo, detailed in vitro studies are needed to characterize thermal tolerance of the fungus without complexities introduced by the species-specific characteristics of hosts' immune systems. The aim of our study was to evaluate the thermal tolerance of a hypervirulent isolate of Bd, considering the limits of its thermal tolerance and its capacity to rebound following heat treatment. We incubated Bd cell cultures at five different temperatures (21, 25.5, 27, 29, or 30.5 C) for one of six exposure durations (3, 4, 5, 6, 7, or 8 days) and subsequently counted the number of zoospores to assess the temperature dependence of Bd growth. We observed intensive Bd growth at 21 C. At 25.5 C, the number of zoospores also increased over time, but the curve plateaued at about half of the maximum values observed in the lower temperature treatment. At temperatures of 27 C and above, the fungus showed no measurable growth. However, when we moved the cultures back to 21 C after the elevated temperature treatments, we observed recovery of Bd growth in all cultures previously treated at 27 C. At 29 C, a treatment duration of 8 days was necessary to prevent recovery of Bd growth, and at 30.5 C a treatment duration of 5 days was needed to achieve the same result, revealing that these moderately elevated temperatures applied for only a few days have merely a fungistatic rather than a fungicidal effect under in vitro conditions.

"Heat waves" experienced during larval life have species-specific consequences on life-history traits and sexual development in anuran amphibians.

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 minimize 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 2 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.

Sex reversal and ontogeny under climate change and chemical pollution: are there interactions between the effects of elevated temperature and a xenoestrogen on early development in agile frogs?

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.

Exposure to Batrachochytrium dendrobatidis affects chemical defences in two anuran amphibians, Rana dalmatina and Bufo bufo.

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.

Host-multiparasite interactions in amphibians: a review.

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.

How to disinfect anuran eggs? Sensitivity of anuran embryos to chemicals widely used for the disinfection of larval and post-metamorphic amphibians.

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.

Relationships Between Chemical Defenses of Common Toad (Bufo bufo) Tadpoles and Bacterial Community Structure of their Natural Aquatic Habitat.

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.

Standardize or Diversify Experimental Conditions in Ecotoxicology? A Case Study on Herbicide Toxicity to Larvae of Two Anuran Amphibians.

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.

Age-dependent changes in sensitivity to a pesticide in tadpoles of the common toad (Bufo bufo).

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.

Effects of a glyphosate-based herbicide and predation threat on the behaviour of agile frog tadpoles.

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 6.5mg 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.

Variation in Chemical Defense Among Natural Populations of Common Toad, Bufo bufo, Tadpoles: the Role of Environmental Factors.

Defensive toxins are widespread in nature, yet we know little about how various environmental factors shape the evolution of chemical defense, especially in vertebrates. In this study we investigated the natural variation in the amount and composition of bufadienolide toxins, and the relative importance of ecological factors in predicting that variation, in larvae of the common toad, Bufo bufo, an amphibian that produces toxins de novo. We found that tadpoles' toxin content varied markedly among populations, and the number of compounds per tadpole also differed between two geographical regions. The most consistent predictor of toxicity was the strength of competition, indicating that tadpoles produced more compounds and larger amounts of toxins when coexisting with more competitors. Additionally, tadpoles tended to contain larger concentrations of bufadienolides in ponds that were less prone to desiccation, suggesting that the costs of toxin production can only be afforded by tadpoles that do not need to drastically speed up their development. Interestingly, this trade-off was not alleviated by higher food abundance, as periphyton biomass had negligible effect on chemical defense. Even more surprisingly, we found no evidence that higher predation risk enhances chemical defenses, suggesting that low predictability of predation risk and high mortality cost of low toxicity might select for constitutive expression of chemical defense irrespective of the actual level of predation risk. Our findings highlight that the variation in chemical defense may be influenced by environmental heterogeneity in both the need for, and constraints on, toxicity as predicted by optimal defense theory.

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

The heavy application of pesticides and its potential effects on natural communities has attracted increasing attention to inadvertent impacts of these chemicals. Toxicologists conventionally use laboratory-based tests to assess lethal concentrations of pesticides. However, these tests often do not take into account indirect, interactive and long-term effects, and tend to ignore different rates of disintegration in the laboratory and under natural conditions. Our aim was to investigate the importance of the experimental venue for ecotoxicology tests. We reared tadpoles of the agile frog (Rana dalmatina) in the laboratory and in outdoor mesocosms and exposed them to three initial concentrations of a glyphosate-based herbicide (0, 2 and 6.5 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.

No observable effect of a glyphosate-based herbicide on two top predators of temporal water bodies.

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.

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

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