Biology subjects

Miko, Z.

Publications and source records attributed to Miko, Z..

11 recordsLinked to original sources

bioRxiv2026

Microplastics disrupt adaptive predator-induced behavioural plasticity in Rana dalmatina tadpoles

The accumulation of microplastics (MP; plastic particles 1 m - 1 mm in diameter) in the environment is an increasing global concern. Although the physiological effects of MP on organisms, including humans, are increasingly documented, their impacts on behaviour are far less understood. Further, little is known about how MP affect adaptive phenotypic plasticity - the ability of a genotype to adaptively modify its phenotype in response to environmental cues. Anuran tadpoles are key models for studying phenotypic plasticity, with well-established evidence for predator-induced behavioural adjustments. Tadpoles typically reduce their activity and risk-taking when exposed to chemical cues released by predators, which has been proven to be adaptive. We investigated whether MP exposure from the fertilised egg stage alters the behaviour of agile frog (Rana dalmatina) tadpoles, and whether it interferes with their predator-induced behavioural plasticity. Tadpoles exposed to chemical cues from dragonfly larvae showed the expected antipredator response: these larvae showed significantly reduced movement activity and risk-taking. Although exposure to MP did not influence the behaviour of tadpoles that had not been exposed to predator cues, and also did not alter predator-induced changes in movement activity, it entirely abolished the predator-induced reduction in risk-taking. These results indicate that MP can compromise antipredator behaviour, increasing the vulnerability of individuals and natural populations to predation without causing visible developmental abnormalities. We recommend that future MP research targets behavioural traits with direct relevance to survival and reproduction, and examines how adaptive phenotypic plasticity is affected.

animal behavior and cognition↗

bioRxiv2025

Photo-based individual identification is more reliable than visible implant elastomer tags or toe clipping in young agile frogs

In amphibian capture-recapture studies, commonly used individual-identification methods include toe clipping as well as less invasive alternatives of varying cost. Yet, choosing the best method for a study is challenging, because both the reliability for identification and the severity of adverse effects of a given mark type can greatly vary between species as well as life stages. We compared the reliability of three identification methods in young agile frogs (Rana dalmatina): clipping a single phalanx, injecting visible-implant-elastomer (VIE) tags (one of six colours) under the skin, and photo-based identification using natural colouration. Individuals were regularly photographed from the start of metamorphosis onwards, and were marked by the other two methods soon after all of them finished metamorphosis. Subsequently, we checked mark retention by each method multiple times for more than a year. Photo-based identification was far more reliable than the other two methods: 100% identification success post-metamorphosis in all checking events within small housing groups, and 98% with computer-assisted identification across all housing groups. Post-metamorphic body colouration remained largely stable, and the major patterns were already present at metamorphosis. Based on VIE colour alone, we could correctly identify the animals in 77% of the checks. Green tags were the easiest to recognize. VIE often broke into smaller parts, and those were sometimes found only outside the originally tagged body part (15%) or not found at all (10%). VIE was better retained in the legs than in the arms. Dissection revealed that migrating VIE pieces can accumulate in the internal organs, especially the kidneys. The clipped toe was successfully recognized only in 41% of checks. We conclude that photo-based identification is superior to both VIE-tagging and toe tipping in young agile frogs, and this method should be preferred in future studies seeking a cost-efficient yet reliable identification method in this species.

ecology↗

bioRxiv2025

Put the frog in water: simple methods for improving individual identification as demonstrated with the agile frog

Identification of individuals across time and space is required for investigating numerous evolutionary-ecology and conservation-related questions, and photo-based identification is commonly used for a broad taxonomic range. Systematic comparisons of available photo-matching software have been published for various taxa, and success of such software may greatly depend on image quality as well as the focal body parts. Yet, suitability assessments of images captured with different methods are missing. Here we tested the hypotheses that individual identification by colour patterns can be facilitated by taking into account the natural medium surrounding the animals and the natural body posture they tend to take. We developed new photography methods to enable individual identification by whole-body assessment using the agile frog (Rana dalmatina), a species with common yet so-far neglected melanin-based pattern on its limbs and back. We compared the reliability of different photography methods for computer-assisted identification in the HotSpotter software as well as for observers operating it. We found that photographing either hand-restrained frogs with towel-dried skin, or frogs moving freely in clean water enabled comparison of the dorsal surface of the whole body including the hind legs, and HotSpotter identified matching images at rates similar to the relatively more successful of previously published anuran studies (>92% listed with rank[≤]10). By contrast, submerging hand-restrained frogs in water significantly improved identification: images of re-captured individuals were always ranked as the most likely match. We attribute this outstanding performance to the combination of advantageous effects of in-water light refractions that improve the visibility of melanin patterns, and uniform body postures facilitating comparison across individuals. Observers in general successfully identified matching images and ruled out non-matching ones, but some mistakes were recorded when images featured freely moving frogs. The photography methods developed in this study should be easily adapted to most frog and toad species for reliable individual identification. Our study highlights that taking into account features of the natural environment of the studied species can improve individual identification by photographs. Because such methods are non-invasive and inexpensive, they should be especially beneficial for population monitoring programs of endangered species. Data availabilityData are available from the FigShare Repository [DOI will be assigned upon acceptance for publication]. Further supporting information for methods and results is provided as Supplement to this manuscript.

ecology↗

bioRxiv2024

Do pyrethroid insecticides influence sex ratios in agile frogs (Rana dalmatina)?

Environmental pollutants have the potential to alter sex ratios in wildlife through sex-biased mortality. Furthermore, endocrine disruptors may cause sex reversal during early ontogeny in ectothermic vertebrates, resulting in a phenotypic sex that is not concordant with the genotypic sex encoded by the sex chromosomes. Despite the wide-ranging implications of these sex-ratio biasing effects, they are rarely studied in ecotoxicology, especially in a way that allows for disentangling the two mechanisms. We investigated these effects of two synthetic pyrethroids, deltamethrin and etofenprox, that are commonly used insecticides and have been linked to adverse effects on fish and amphibian biodiversity. We assessed the effects of environmentally relevant concentrations of these two pyrethroids on phenotypic sex ratio, sex-dependent mortality, and sex reversal in agile frogs (Rana dalmatina). Tadpoles from field-collected eggs were reared in mesocosm until metamorphosis by adding 0.03 or 0.3 g/L of deltamethrin or etofenprox three times to the water. We observed no effect in three of the four treatment groups. However, in the lower-concentration etofenprox treatment, phenotypic sex ratio was male-biased two months post-metamorphosis, and genotypic sexing revealed that this was due to female-biased mortality during metamorphosis and not to sex reversal. Although the estimation certainty of these effects was somewhat limited, they highlight that not all sex-ratio distorting effects are caused by sex reversal. Therefore, ecotoxicological studies aiming to understand the endocrine distruptor effects of environmental contaminants should strive to separate the effects on sex determination and sex-dependent mortality.

ecology↗

bioRxiv2024

Juvenile agile frogs spatially avoid ranavirus-infected conspecifics, but do not show generalized social distancing or self-isolation

Exposure to contagious pathogens can result in behavioural changes, which can alter the spread of infectious diseases. Healthy individuals can express generalized social distancing or actively avoid the sources of infection, while infected individuals can show passive or active self-isolation. Amphibians are globally threatened by serious contagious diseases, yet their behavioural responses to infections are very scarcely known. We studied behavioural changes in agile frog (Rana dalmatina) juveniles upon exposure to a Ranavirus (Rv). We performed classic choice tests in chambers containing a conspecific infected with Rv in one end compartment and a non-infected conspecific in the other end, with an Rv-infected or non-infected focal individual in the central compartment. We found that both non-infected and Rv- infected focal individuals spatially avoided infected conspecifics, while there were no signs of generalized social distancing, nor self-isolation. Spatial avoidance of infected conspecifics may effectively hinder disease transmission. On the other hand, the absence of self-isolation by infected individuals may facilitate it. Our finding that infected individuals spent more time near the non-infected than infected conspecifics suggests that the strong behavioural drive to avoid infected conspecifics may not be silenced by infection, possibly to prevent secondary infections. The observation that infected focal individuals did not spend more time near conspecifics than non-infected focals renders it unlikely that the pathogen manipulated host behaviour to aid disease spread. More research is urgently needed to understand under what circumstances behavioural responses can help amphibians cope with infections, and how that affects disease dynamics in natural populations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/588850v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@59b5a9org.highwire.dtl.DTLVardef@1a59896org.highwire.dtl.DTLVardef@c4bc64org.highwire.dtl.DTLVardef@2c76c3_HPS_FORMAT_FIGEXP M_FIG C_FIG

animal behavior and cognition↗

bioRxiv2023

Effects of mild winter conditions and two pyrethroid insecticides on the development and survival of juvenile common toads (Bufo bufo)

Combined effects of pollutants and climate change can have long-term negative effects on Earths biodiversity. We tested how amphibians are affected by exposure to pesticides around metamorphosis in combination with current winter conditions or those projected to become the norm by 2100. We applied two pyrethroid insecticides, cypermethrin or deltamethrin, to newly metamorphosed common toads (Bufo bufo) via spraying, thereafter reared animals in outdoor enclosures, and finally exposed them either to a long&cold winter (1.5 {degrees}C for 91 days) or to a short&mild one (4.5 {degrees}C for 61 days). After hibernation, we examined changes in body mass, size and gross morphology of parotoid glands and visceral organs, and determined the phenotypic sex of the surviving toadlets. In the long&cold winter scenario, we observed high mortality rate, regardless of the chemical treatment (control: 82%, cypermethrin: 78%, deltamethrin: 70%). At the same time, mortality was significantly lower in the short&mild winter scenario (control: 33%, cypermethrin: 41%, deltamethrin: 26%). The decrease in body mass during overwintering did not differ among treatment groups. Our results contradict the hypothesis of sex-biased mortality caused by chemical pollutants, harsh or mild winter conditions or by their combination. No significant deformations of the internal organs were found in either group. Our results suggest that winter conditions becoming ever milder in the temperate zone may prove beneficial for amphibian overwinter survival, and that exposure to the tested pyrethroids at ecologically relevant concentrations after metamorphosis does not interfere with the fitness of juvenile common toads.

zoology↗

bioRxiv2023

Infection with the chytrid fungus (Batrachochytrium dendrobatidis) may distort sex ratio via sex reversal but not sex-dependent mortality in a common Eurasian amphibian

One of the major factors driving the currently ongoing biodiversity crisis is the anthropogenic spread of infectious diseases. Diseases can have conspicuous consequences, such as mass mortality events, but may also exert covert but similarly severe effects, such as sex ratio distortion via sex-biased mortality or sex reversal. Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is among the most important threats to amphibian biodiversity. Yet, whether Bd infection can skew sex ratios in amphibians is currently unknown, although such a hidden effect may cause the already dwindling amphibian populations to collapse. To investigate this possibility, we collected common toad (Bufo bufo) tadpoles from a natural habitat in Hungary, and continuously treated them until metamorphosis with sterile Bd culture medium (control), or a liquid culture of a Hungarian or a Spanish Bd isolate. Three months after metamorphosis we dissected the individuals and diagnosed their phenotypic and genetic sex. Bd prevalence was high in animals that died during the experiment but was almost zero at sexing. Survival was generally low in the control group, but it was further lowered by the Bd treatments. We did not observe sex-dependent mortality in either treatment. However, treatment with the Spanish Bd isolate significantly increased the frequency of sex reversal: 3 out of 9 genetic females developed into phenotypic males. Based on our results, Bd infection may have the potential to affect sex ratio in common toads through female-to-male sex reversal, but future research is needed to ascertain the generality of these findings.

developmental biology↗

bioRxiv2023

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.

physiology↗

bioRxiv2021

"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 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 {degrees}C and exposed them to a simulated heat wave of 28 or 30 {degrees}C for six days during one of three ontogenetic periods (early, mid or late larval development). In agile frogs, exposure to 30 {degrees}C during early larval development increased mortality. Regardless of timing, all heat-treatments delayed metamorphosis, and exposure to 30 {degrees}C decreased body mass at metamorphosis. Furthermore, exposure to 30 {degrees}C during any period and to 28 {degrees}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.

ecology↗

bioRxiv2021

Effects of chlorpyrifos on early development and anti-predator behavior of agile frogs

The widespread application of pesticides makes it important to understand the impacts of these chemicals on wildlife populations. Chlorpyrifos is an organophosphate insecticide which can affect the development and behavior of aquatic organisms and may thereby alter predator-prey interactions. To investigate how environmentally relevant, sublethal concentrations of chlorpyrifos affect anti-predator behavior and larval development of the agile frog (Rana dalmatina), we exposed tadpoles to one of three concentrations (0, 0.5 and 5 g chlorpyrifos / L) either for a brief period of three days (acute exposure) or throughout larval development (chronic exposure). We observed tadpole activity and space use in the presence or absence of chemical cues of predatory fish. We also assessed mortality, time to metamorphosis, mass at metamorphosis, brain morphology and sex ratio. We found that tadpoles chronically exposed to 5 g/L chlorpyrifos swam distances that were longer by more than 20 % and exhibited body masses at metamorphosis that were lower by ca. 7 % than in control individuals, but the other fitness-related traits remained unaffected. The lower concentration of chlorpyrifos applied chronically, and either one of the acute chlorpyrifos treatments did not influence any measured trait. Our results demonstrate that exposure to chlorpyrifos can induce changes in locomotor activity and may result in lowered body mass of agile frog tadpoles, but only if the insecticide is present chronically at concentrations which are rarely reached in natural waterbodies. Thus, agile frog tadpoles appear to be relatively tolerant to chlorpyrifos, but may nonetheless suffer from its presence in situations of repeated high-dose application.

ecology↗

bioRxiv2020

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 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 (masculinizing agent) and 17-ethinylestradiol (EE2), a widespread xenoestrogen (feminizing agent), on sexual development and fitness-related traits in agile frogs (Rana dalmatina). We exposed tadpoles to a six-days heat wave (30 {degrees}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 masculinizing 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.

pharmacology and toxicology↗