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

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

Comprehensive analyses of the occurrence of a fungicide resistance marker and the genetic structure in Erysiphe necator populations.

Genetically distinct groups of Erysiphe necator, the fungus causing grapevine powdery mildew infect grapevine in Europe, yet the processes sustaining stable genetic differences between those groups are less understood. Genotyping of over 2000 field samples from six wine regions in Hungary collected between 2017 and 2019 was conducted to reveal E. necator genotypes and their possible differentiation. The demethylase inhibitor (DMI) fungicide resistance marker A495T was detected in all wine regions, in 16% of the samples. Its occurrence differed significantly among wine regions and grape cultivars, and sampling years, but it did not differ between DMI-treated and untreated fields. Multilocus sequence analyses of field samples and 59 in vitro maintained isolates revealed significant genetic differences among populations from distinct wine regions. We identified 14 E. necator genotypes, of which eight were previously unknown. In contrast to the previous concept of A and B groups, European E. necator populations should be considered genetically more complex. Isolation by geographic distance, growing season, and host variety influence the genetic structuring of E. necator, which should be considered both during diagnoses and when effective treatments are planned.

HerpSexDet: the herpetological database of sex determination and sex reversal.

Wildlife exhibits various sex-determination systems where sex chromosomes and environmental temperatures may both contribute to individual sexual development. The causes and consequences of this variability are important questions for evolutionary ecology, especially in light of ongoing environmental change. Amphibians and reptiles are emerging as a key group for studying these questions, with new data accumulating acceleratingly. We collected empirical data from earlier databases, reviews and primary literature to create the most up-to-date database on herpetological sex determination. We named our database HerpSexDet, which currently features data on genetic and temperature-dependent sex determination as well as reports on sex reversal for a total of 192 amphibian and 697 reptile species. This dataset, which we will regularly update in the future, facilitates interspecific comparative studies on the evolution of sex determination and its consequences for species-specific traits such as life history and conservation status, and may also help guiding future research by identifying species or higher taxa that are potentially most enlightening for the study of environmentally driven sex reversal.

Multiple paternity is related to adult sex ratio and sex determination system in reptiles.

The adult sex ratio (ASR, the proportion of males in the adult population) is an emerging predictor of reproductive behaviour, and recent studies in birds and humans suggest it is a major driver of social mating systems and parental care. ASR may also influence genetic mating systems. For instance male-skewed ASRs are expected to increase the frequency of multiple paternity (defined here as a clutch or litter sired by two or more males) due to higher rates of coercive copulations by males, and/or due to females exploiting the opportunity of copulation with multiple males to increase genetic diversity of their offspring. Here, we evaluate this hypothesis in reptiles that often exhibit high frequency of multiple paternity although its ecological and life-history predictors have remained controversial. Using a comprehensive dataset of 81 species representing all four non-avian reptile orders, we show that increased frequency of multiple paternity is predicted by more male-skewed ASR, and this relationship is robust to simultaneous effects of several life-history predictors. Additionally, we show that the frequency of multiple paternity varies with the sex determination system: species with female heterogamety (ZZ/ZW sex chromosomes) exhibit higher levels of multiple paternity than species with male heterogamety (XY/XX) or temperature-dependent sex determination. Thus, our across-species comparative study provides the first evidence that genetic mating system depends on ASR in reptiles. We call for further investigations to uncover the complex evolutionary associations between mating systems, sex determination systems and ASR.

Oxidative physiology is weakly associated with pigmentation in birds.

The mechanistic link between avian oxidative physiology and plumage coloration has attracted considerable attention in past decades. Hence, multiple proximal hypotheses were proposed to explain how oxidative state might covary with the production of melanin and carotenoid pigments. Some hypotheses underscore that these pigments (or their precursors, e.g., glutathione) have antioxidant capacities or function as molecules storing the toxic excess of intracellular compounds, while others highlight that these pigments can act as pro-oxidants under specific conditions. Most studies addressing these associations are at the intraspecific level, while phylogenetic comparative studies are still scarce, though needed to assess the generality of these associations. Here, we tested whether plumage and bare part coloration were related to oxidative physiology at an interspecific level by measuring five oxidative physiology markers (three nonenzymatic antioxidants and two markers of lipid peroxidative damage) in 1387 individuals of 104 European bird species sampled during the breeding season, and by scoring plumage eumelanin, pheomelanin, and carotenoid content for each sex and species. Only the plasma level of reactive oxygen metabolites was related to melanin coloration, being positively associated with eumelanin score and negatively with pheomelanin score. Thus, our results do not support the role of antioxidant glutathione in driving variation in melanin synthesis across species. Furthermore, the carotenoid scores of feathers and bare parts were unrelated to the measured oxidative physiology parameters, further suggesting that the marked differences in pigmentation across birds does not influence their oxidative state.

Asymmetrical sex reversal: Does the type of heterogamety predict propensity for sex reversal?

Sex reversal, a mismatch between phenotypic and genetic sex, can be induced by chemical and thermal insults in ectotherms. Therefore, climate change and environmental pollution may increase sex-reversal frequency in wild populations, with wide-ranging implications for sex ratios, population dynamics, and the evolution of sex determination. We propose that reconsidering the half-century old theory "Witschi's rule" should facilitate understanding the differences between species in sex-reversal propensity and thereby predicting their vulnerability to anthropogenic environmental change. The idea is that sex reversal should be asymmetrical: more likely to occur in the homogametic sex, assuming that sex-reversed heterogametic individuals would produce new genotypes with reduced fitness. A review of the existing evidence shows that while sex reversal can be induced in both homogametic and heterogametic individuals, the latter seem to require stronger stimuli in several cases. We provide guidelines for future studies on sex reversal to facilitate data comparability and reliability.

"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.

Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats.

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable because environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex-ratio distortion and population decline. Despite these implications, there is scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex-reversing stimuli more frequently, which may lead to higher sex-reversal rate or, alternatively, these populations may adapt to resist sex reversal. We developed PCR-based genetic sex markers for the common toad (Bufo bufo) to assess the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous oestrogenic pollutants in a common garden experiment. We found negligible sex-reversal frequency in free-living adults despite the presence of various endocrine-disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg L-1 17α-ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng L-1 EE2 and a glyphosate-based herbicide with 3 µg L-1 or 3 mg L-1  glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the extremely low sex-reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species-specific resistance to sex reversal.

Coping With Urban Habitats Via Glucocorticoid Regulation: Physiology, Behavior, and Life History in Stream Fishes.

As environments become urbanized, tolerant species become more prevalent. The physiological, behavioral and life-history mechanisms associated with the success of such species in urbanized habitats are not well understood, especially in freshwater ecosystems. Here, we examined the glucocorticoid (GC) profiles, life-history traits, and behavior of two species of fish across a gradient of urbanization to understand coping capacity and associated trade-offs. We studied the tolerant live-bearing Western Mosquitofish (Gambusia affinis) for two years and the slightly less tolerant, egg-laying, Blacktail Shiner (Cyprinella venusta) for one year. We used a water-borne hormone method to examine baseline, stress-induced, and recovery cortisol release rates across six streams with differing degrees of urbanization. We also measured life-history traits related to reproduction, and for G. affinis, we measured shoaling behavior and individual activity in a novel arena. Both species showed a trend for reduced stress responsiveness in more urbanized streams, accompanied by higher reproductive output. Although not all populations fit this trend, these results suggest that GC suppression may be adaptive for coping with urban habitats. In G. affinis, GC recovery increased with urbanization, and individuals with the lowest stress response and highest recovery had the greatest reproductive allotment, suggesting that rapid return to baseline GC levels is also an important coping mechanism. In G. affinis, urban populations showed altered life-history trade-offs whereas behavioral traits did not vary systematically with urbanization. Thus, these tolerant species of fish may cope with anthropogenically modified streams by altering their GC profiles and life-history trade-offs. These results contribute to understanding the mechanisms driving species-specific adaptations and thereby community structure in freshwater systems associated with land-use converted areas.

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.

Evolution of large males is associated with female-skewed adult sex ratios in amniotes.

Body size often differs between the sexes (leading to sexual size dimorphism, SSD), as a consequence of differential responses by males and females to selection pressures. Adult sex ratio (ASR, the proportion of males in the adult population) should influence SSD because ASR relates to both the number of competitors and available mates, which shape the intensity of mating competition and thereby promotes SSD evolution. However, whether ASR correlates with SSD variation among species has not been yet tested across a broad range of taxa. Using phylogenetic comparative analyses of 462 amniotes (i.e., reptiles, birds, and mammals), we fill this knowledge gap by showing that male bias in SSD increases with increasingly female-skewed ASRs in both mammals and birds. This relationship is not explained by the higher mortality of the larger sex because SSD is not associated with sex differences in either juvenile or adult mortality. Phylogenetic path analysis indicates that higher mortality in one sex leads to skewed ASR, which in turn may generate selection for SSD biased toward the rare sex. Taken together, our findings provide evidence that skewed ASRs in amniote populations can result in the rarer sex evolving large size to capitalize on enhanced mating opportunities.

Factors modulating the behavioral and physiological stress responses: Do they modify the relationship between flight initiation distance and corticosterone reactivity?

Understanding how vulnerable species are to new stressors, such as anthropogenic changes, is crucial for mitigating their potential negative consequences. Many studies have investigated species sensitivity to human disturbance by focusing on single behavioral or physiological parameters, such as flight initiation distance and glucocorticoid levels. However, little is known about the differential effect that modulating factors might have on behavioral versus physiological stress responses across species. This lack of knowledge make difficult to understand the relationship between both types of reactions, and thus to assess to what extent a behavioral reaction is representative of an internal physiological stress response or vice versa. We collected published data on bird flight initiation distances (FID) and corticosterone (CORT) responses, the two most frequently used indicators of stress reaction. We then investigated how spatio-temporal factors or species-specific characteristics relate to these behavioral and physiological stress responses, and potentially modify the relationship between them. Additionally, we evaluated the strength of the correlation between the two stress responses (behavioral and physiological). Our findings showed that FID and CORT responses were poorly correlated across species, and the lack of correlation was attributable to modulating factors (e.g. latitude and body mass) which influence behavior and physiology differently. These modulating factors, therefore, should be taken into consideration to better interpret FID and CORT responses in the context of species vulnerability to stress.

Evolutionary and demographic consequences of temperature-induced masculinization under climate warming: the effects of mate choice.

BACKGROUND: One of the dangers of global climate change to wildlife is distorting sex ratios by temperature-induced sex reversals in populations where sex determination is not exclusively genetic, potentially leading to population collapse and/or sex-determination system transformation. Here we introduce a new concept on how these outcomes may be altered by mate choice if sex-chromosome-linked phenotypic traits allow females to choose between normal and sex-reversed (genetically female) males. RESULTS: We developed a theoretical model to investigate if an already existing autosomal allele encoding preference for sex-reversed males would spread and affect demographic and evolutionary processes under climate warming. We found that preference for sex-reversed males (1) more likely spread in ZW/ZZ than in XX/XY sex-determination systems, (2) in populations starting with ZW/ZZ system, it significantly hastened the transitions between different sex-determination systems and maintained more balanced adult sex ratio for longer compared to populations where all females preferred normal males; and (3) in ZW/ZZ systems with low but non-zero viability of WW individuals, a widespread preference for sex-reversed males saved the populations from early extinction. CONCLUSIONS: Our results suggest that climate change may affect the evolution of mate choice, which in turn may influence the evolution of sex-determination systems, sex ratios, and thereby adaptive potential and population persistence. These findings show that preferences for sex-linked traits have special implications in species with sex reversal, highlighting the need for empirical research on the role of sex reversal in mate choice.

Are evolutionary transitions in sexual size dimorphism related to sex determination in reptiles?

Sex determination systems are highly variable in vertebrates, although neither the causes nor the implications of this diversity are fully understood. Theory suggests that sex determination is expected to relate to sexual size dimorphism, because environmental sex determination promotes sex-specific developmental bias in embryonic growth rates. Furthermore, selection for larger size in one sex or the other has been proposed to drive the evolution of different genetic sex determination systems. Here, we investigate whether sex determination systems relate to adult sexual size dimorphism, using 250 species of reptiles (Squamata, Testudines and Crocodylia) representing 26 families. Using phylogenetically informed analyses, we find that sexual size dimorphism is associated with sex determination: species with TSDIa sex determination (i.e. in which the proportion of female offspring increases with incubation temperature) have more female-biased size dimorphism than species with TSDII (i.e. species in which males are produced at mid temperatures). We also found a trend that species with TSD ancestors had more male-biased size dimorphism in XY sex chromosome systems than in ZW sex chromosome systems. Taken together, our results support the prediction that sexual size dimorphism is linked to sex-dependent developmental variations caused by environmental factors and also by sex chromosomes. Since the extent of size dimorphism is related to various behavioural, ecological and life-history differences between sexes, our results imply profound impacts of sex determination systems for vertebrate diversity.

Stressed tadpoles mount more efficient glucocorticoid negative feedback in anthropogenic habitats due to phenotypic plasticity.

Coping with anthropogenic environmental change is among the greatest challenges faced by wildlife, and endocrine flexibility is a potentially crucial coping mechanism. Animals may adapt to anthropogenic environments by dampening their glucocorticoid stress response, but empirical tests of this hypothesis have provided mixed evidence. An alternative hypothesis is that a non-attenuated stress response and efficient negative feedback are favored in anthropogenic habitats. To test this idea, we non-invasively sampled corticosterone release rates of common toad (Bufo bufo) tadpoles in agricultural, urban, and natural habitats, and quantified their stress response and negative feedback by a standardized stress-and-recovery protocol. We repeated the same sampling with tadpoles raised from eggs from the same ponds in a common-garden experiment to infer if the differences observed between populations in different habitats were due to individual phenotypic plasticity rather than microevolution or transgenerational effects. We found that, compared to tadpoles in natural ponds, urban tadpoles had higher baseline and stressed corticosterone release rates, and tadpoles in agricultural ponds had similar corticosterone release rates but greater stress-induced change, indicating stronger stress responses in both types of anthropogenic habitats. As predicted, tadpoles in both agricultural and urban ponds showed more efficient negative feedback than did tadpoles in natural ponds. Water pollution levels, as indicated by the concentrations of carbamazepine and corticoid-disrupting compounds in pond water, contributed to elevating the stress response regardless of land use. Infection by neither Batrachochytrium dendrobatidis nor Ranavirus was detected in free-living tadpoles. No habitat-related glucocorticoid differences persisted in the common-garden experiment. These results suggest that toad tadpoles in anthropogenic habitats increased their glucocorticoid flexibility via phenotypic plasticity. The coupling of stronger stress response and stronger negative feedback in these habitats supports the importance of rapidly "turning on and off" the stress response as a mechanism for coping with anthropogenic environmental change.

Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use.

Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex-linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex-reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress-induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North-Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female-to-male sex-reversed adults had similar body mass as normal males. We recorded no events of male-to-female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human-induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex-reversed individuals surviving to adulthood may participate in breeding.

Effects of two little-studied environmental pollutants on early development in anurans.

Despite intensive ecotoxicological research, we still know relatively little about the ecological impacts of many environmental contaminants. Filling these knowledge gaps is particularly important regarding amphibians, because they play significant roles in freshwater and terrestrial ecosystems, and their populations are declining worldwide. In this study, we investigated two pollutants that have been poorly studied in ecotoxicology despite their widespread occurrence in surface waters: the herbicide terbuthylazine and the pharmaceutical drug carbamazepine. We exposed two anuran species throughout their larval development to each of two environmentally relevant concentrations of each pollutant, and recorded mortality and 17 sub-lethal endpoints up to several months after exposure. Mortality was low and unrelated to treatment. In agile frogs (Rana dalmatina), we found that treatment with 0.3 µg/L terbuthylazine decreased tadpole activity and reduced fat bodies in juveniles, whereas treatment with 50 µg/L carbamazepine decreased spleen size and increased spleen pigmentation. In common toads (Bufo bufo), treatment with 0.003 µg/L terbuthylazine increased body mass at metamorphosis, treatment with 0.3 µg/L terbuthylazine increased the size of optic tecta, and treatment with 0.5 µg/L carbamazepine decreased hypothalamus size. Treatment with 50 µg/L carbamazepine reduced the feeding activity of toad tadpoles, decreased their production of anti-predatory bufadienolide toxins, and increased their body mass at metamorphosis; juvenile toads in this treatment group had reduced spleen pigmentation. Neither treatments affected the time to metamorphosis, post-metamorphic body mass, or sex ratios significantly. These results show that environmental levels of both terbuthylazine and carbamazepine can have several sub-lethal effects on anurans, which may be detrimental to individual fitness and population persistence in natural conditions. Our findings further highlight that toxic effects cannot be generalized between chemicals of similar structure, because the terbuthylazine effects we found do not conform with previously reported effects of atrazine, a related and extensively studied herbicide.

Does offspring sex ratio differ between urban and forest populations of great tits (Parus major)?

Since male and female offspring may have different costs and benefits, parents may use sex ratio adjustment to increase their own fitness under different environmental conditions. Urban habitats provide poorer conditions for nestling development in many birds. Therefore, we investigated whether great tits (Parus major) produce different brood sex ratios in urban and natural habitats. We determined the sex of nestlings of 126 broods in two urban and two forest sites between 2012 and 2014 by molecular sexing. We found that brood sex ratio did not differ significantly between urban and forest habitats either at egg-laying or near fledging. Male offspring were larger than females in both habitats. This latter result suggests that male offspring may be more costly to raise than females, yet our findings suggest that urban great tits do not produce more daughters despite the unfavourable breeding conditions. This raises the possibility that other aspects of urban life, such as better post-fledging survival, might favour males and thereby compensate for the extra energetic costs of producing male offspring.

Predator-induced changes in the chemical defence of a vertebrate.

1. Inducible defences are ubiquitous in the animal kingdom, but little is known about facultative changes in chemical defences in response to predators, especially so in vertebrates. 2. We tested for predator-induced changes in toxin production of larval common toads (Bufo bufo), which are known to synthesize bufadienolide compounds. 3. The experiment included larvae originating from three permanent and three temporary ponds reared in the presence or absence of chemical cues of three predators: dragonfly larvae, newts or fish. 4. Tadpoles raised with chemical cues of predation risk produced higher numbers of bufadienolide compounds and larger total bufadienolide quantities than predator-naive conspecifics. Further, the increase in intensity of chemical defence was greatest in response to fish, weakest to newts and intermediate to dragonfly larvae. Tadpoles originating from temporary and permanent ponds did not differ in their baseline toxin content or in the magnitude of their induced chemical responses. 5. These results provide the first compelling evidence for predator-induced changes in chemical defence of a vertebrate that may have evolved to enhance survival under predation risk.

Chemical defense of toad tadpoles under risk by four predator species.

Many organisms use inducible defenses as protection against predators. In animals, inducible defenses may manifest as changes in behavior, morphology, physiology, or life history, and prey species can adjust their defensive responses based on the dangerousness of predators. Analogously, prey may also change the composition and quantity of defensive chemicals when they coexist with different predators, but such predator-induced plasticity in chemical defenses remains elusive in vertebrates. In this study, we investigated whether tadpoles of the common toad (Bufo bufo) adjust their chemical defenses to predation risk in general and specifically to the presence of different predator species; furthermore, we assessed the adaptive value of the induced defense. We reared tadpoles in the presence or absence of one of four caged predator species in a mesocosm experiment, analyzed the composition and quantity of their bufadienolide toxins, and exposed them to free-ranging predators. We found that toad tadpoles did not respond to predation risk by upregulating their bufadienolide synthesis. Fishes and newts consumed only a small percentage of toad tadpoles, suggesting that bufadienolides provided protection against vertebrate predators, irrespective of the rearing environment. Backswimmers consumed toad tadpoles regardless of treatment. Dragonfly larvae were the most voracious predators and consumed more predator-naïve toad tadpoles than tadpoles raised in the presence of dragonfly cues. These results suggest that tadpoles in our experiment had high enough toxin levels for an effective defense against vertebrate predators even in the absence of predator cues. The lack of predator-induced phenotypic plasticity in bufadienolide synthesis may be due to local adaptation for constantly high chemical defense against fishes in the study population and/or due to the high density of conspecifics.