Patterns in parasite diversity and interactions with anurans from the Atlantic Forest.

Knowledge of parasite-host interactions is essential for understanding factors associated with the ecology and evolution of both groups. Some aspects, such as host size and phylogeny, as well as parasite specificity, are significant predictors that help unveil the parasite-host relationship. Thus, the goals of this study were: (1) to describe parasite diversity in regions of the Atlantic Forest; (2) to analyze which host characteristics can influence parasite richness of anuran's parasite component community; and (3) to investigate if the prevalence of parasite infection is related to specificity metrics (ecological and phylogenetic), number of infected hosts and parasite's abundance. We identified 49 parasite taxa, classified into three phyla: Nematoda, Acanthocephala, and Platyhelminthes. Supporting the existing literature, our findings corroborate the positive relationship between host size and parasite richness, further emphasizing the significance of this predictor. Parasite prevalence in the host community is related to the number of infected host species and parasite abundance, but not to phylogenetic and ecological specificity indices. This shows that parasite prevalence is strongly associated with infection opportunity, host sampling effort, and high parasite abundance.

Occurrence of common frog (Rana temporaria) and common toad (Bufo bufo) adults and metamorphs in agricultural fields in Germany: Potential for exposure to plant protection products.

To characterize the potential for exposure of amphibian terrestrial life stages to plant protection products (PPP), we studied the occurrence and habitat use of adult and metamorph common frogs (Rana temporaria) and common toads (Bufo bufo) in an agricultural landscape in Germany. The four selected study sites were breeding ponds with approximately 80% agricultural land within a 1-km radius. Adults were monitored by radio tracking for two years, and metamorph numbers were assessed for one summer using pitfall traps alongside drift fences. The results demonstrate that adults were rarely present in arable fields at any of the sites (overall 0.5% and 4% of total observations for frogs and toads, respectively). Metamorph captures in arable fields were more variable, ranging from 1.2% to 38.8% (frogs) and from 0.0% to 26.1% (toads) across study sites. Unsurprisingly, most captures in arable fields for both toad and frog metamorphs occurred at the site where the pond was completely surrounded by arable fields. Overall, the presence of adult amphibians in arable fields was limited and, for the metamorphs, occurred primarily when crops were denser and PPP spray interception higher. Diurnal hiding behavior was observed with the highest activity recorded at night, further reducing the risk of dermal exposure from direct PPP overspraying. In addition, it appeared that alternative habitats, such as woody structures or water bodies in the broader surrounding area, were preferred by the animals over the arable areas. The use of buffer zones around water bodies in agricultural areas would be an effective risk mitigation measure to protect terrestrial adults and metamorphs residing there and would reduce spray drift entry into water bodies during PPP application. It is hoped that these results will contribute to the discussion of risk assessment and mitigation options for amphibians. Integr Environ Assess Manag 2024;00:1-13. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Impacts of a Simulated Infection on the Locomotor Behavior of Invasive and Noninvasive Species of Congeneric Anurans.

AbstractLocomotion is essential for survival, but it requires resources such as energy and metabolites and therefore may conflict with other physiological processes that also demand resources, particularly expensive processes such as immunological responses. This possible trade-off may impose limits on either the magnitude of immune responses or the patterns of activity and performance. Previous studies have shown that invasive species may have a depressed immune response, allowing them to maintain locomotor function and reproduction even when sick. This may contribute to the ecological success of invasive species in colonization and dispersal. In contrast, noninvasive species tend to reduce activity as a response to infection. Here, we studied the impact of a simulated infection on locomotor performance and voluntary movement in the anurans Xenopus laevis (a globally invasive species) and Xenopus allofraseri (a noninvasive congeneric). We found that a simulated infection reduces locomotor performance in both species, with an accentuated effect on X. allofraseri. Voluntary movement was marginally different between species. Our data suggest that a simulated infection leads to behavioral depression and reduced locomotor performance in anurans and show that this effect is limited in the invasive X. laevis. Contrasting responses to an immune challenge have been reported in the few amphibian taxa analyzed to date and suggest relationships between ecology and immunology that deserve further investigation. Specifically, a depressed immune response may underlie a propension to invasion in some species. Whether this is a general trend for invasive species remains to be tested, but our data add to the growing body of work documenting depressed immune systems in invasive species.

Gluconeogenesis in frogs during cooling and dehydration exposure: new insights into tissue plasticity of the gluconeogenic pathway dependent on abiotic factors.

Anurans undergo significant physiological changes when exposed to environmental stressors such as low temperatures and humidity. Energy metabolism and substrate management play a crucial role in their survival success. Therefore, understanding the role of the gluconeogenic pathway and demonstrating its existence in amphibians is essential. In this study, we exposed the subtropical frog Boana pulchella to cooling (-2.5°C for 24 h) and dehydration conditions (40% of body water loss), followed by recovery (24 h), and assessed gluconeogenesis activity from alanine, lactate, glycerol and glutamine in the liver, muscle and kidney. We report for the first time that gluconeogenesis activity by 14C-alanine and 14C-lactate conversion to glucose occurs in the muscle tissue of frogs, and this tissue activity is influenced by environmental conditions. Against the control group, liver gluconeogenesis from 14C-lactate and 14C-glycerol was lower during cooling and recovery (P<0.01), and gluconeogenesis from 14C-glutamine in the kidneys was also lower during cooling (P<0.05). In dehydration exposure, gluconeogenesis from 14C-lactate in the liver was lower during recovery, and that from 14C-alanine in the muscle was lower during dehydration (P<0.05). Moreover, we observed that gluconeogenesis activity and substrate preference respond differently to cold and dehydration. These findings highlight tissue-specific plasticity dependent on the nature of the encountered stressor, offering valuable insights for future studies exploring this plasticity, elucidating the importance of the gluconeogenic pathway and characterizing it in anuran physiology.

Baseline and stress-induced steroid plasma levels and immune function vary annually and are associated with vocal activity in male toads (Rhinella icterica).

Theoretical models predict that elevated androgen and glucocorticoid levels in males during the reproductive season promote immunosuppression. However, some studies report decreased stress response during this season. This study investigated annual variation in plasma corticosterone and testosterone levels, plasma bacterial killing ability (BKA), and neutrophil to lymphocyte ratio (NLR) in free-living male toads (Rhinella icterica). Toads were sampled in the field (baseline) and 1 h-post restraint over five months, and we considered the occurrence of vocal activity. Baseline corticosterone, testosterone, and BKA showed higher values during the reproductive period, specifically in calling male toads. The NLR was similar throughout the year, but higher values were observed in calling toads. Moreover, baseline NLR and BKA were positively correlated with both testosterone and corticosterone, suggesting higher steroid levels during reproduction are associated with enhanced cellular and humoral immunity. Despite fluctuation of baseline values, post-restraint corticosterone levels remained uniform over the year, indicating that toads reached similar maximum values throughout the year. Testosterone levels decreased following restraint before one specific reproductive period but increased in response to restraint during and after this period. Meanwhile, BKA decreased due to restraint only after the reproductive period, indicating immune protection and resilience to immunosuppression by stressors associated with steroid hormones during reproduction. Our results show that baseline and stress-induced hormonal and immune regulation varies throughout the year and are associated with vocal activity in R. icterica males, indicating a possible compromise between steroids and immune function in anuran males.

How do different concentrations of aluminum and zinc affect the survival, body size, morphology and immune system of Physalaemus cuvieri (Fitzinger, 1826) tadpole?

The assessment of amphibian responses as bioindicators of exposure to chemical pollutants is an important tool for conservation of native species. This study aimed to investigate the effects of chronic aluminum (Al) and zinc (Zn) exposure on survival, body size, morphology (malformations), and immune system (leukocyte profile) in P. cuvieri tadpoles. Ecotoxicological analyses were performed utilizing chronic toxicity tests in which 210 tadpoles at the 25th Gosner developmental stage were exposed to Al and Zn. Individuals of P. cuvieri were maintained in glass containers containing various concentrations of aluminum sulfate (0.1, 0.2, or 0.3 mg/L) and zinc sulfate (0.18, 0.27 or 0.35 mg/L), and tests were performed in triplicate. After 14 days, amphibians were weighed, measured and survival rate, malformations in the oral and intestine apparatus, leukocyte profile, and ratio between neutrophils and lymphocytes determined. The differing concentrations of Al and Zn did not produce lethality in P. cuvieri where 95% of the animals survived 326 hr following metal exposure. Individuals exposed to Zn achieved greater body growth and weight gain compared to controls. Aluminum increased weight gain compared controls. These metals also produced malformations of the oral and intestine apparatus and enhanced occurrence of hemorrhages, especially at the highest doses. Lymphocytes were the predominant cells among leukocytes, with lymphopenia and neutrophilia observed following Al and Zn treatment, as evidenced by elevated neutrophil/lymphocyte ratio, an important indicator of stress in animals. Data suggest that further studies need to be carried out, even with metal concentrations higher than those prescribed by CONAMA, to ensure the conservation of this species.

Microplastics and TiO2 nanoparticles mixture as an emerging threat to amphibians: A case study on bullfrog embryos.

Emerging contaminants can act as contributing factors to the decline of amphibian populations worldwide. Recently, scientists have drawn attention to the potential ecotoxicity of microplastics and nanomaterials in amphibians, however, their possible effects on embryonic developmental stages are still absent. Thus, the present study analyzed the developmental toxicity of environmentally relevant concentrations of polyethylene microplastics (PE MPs; 60 mg/L) and titanium dioxide nanoparticles (TiO2 NPs; 10 µg/L), isolated or in combination (Mix group) on bullfrog embryos, Aquarana catesbeiana, adapting the Frog Embryo Teratogenesis Assay (FETAX, 96h). Allied to the FETAX protocol, we also analyzed the heart rate and morphometric data. The exposure reduced the survival and hatching rates in groups exposed to TiO2 NPs, and to a lesser extent, also affected the Mix group. TiO2 NPs possibly interacted with the hatching enzymes of the embryos, preventing hatching, and reducing their survival. The reduced effects in the Mix group are due to the agglomeration of both toxicants, making the NPs less available for the embryos. PE MPs got attached to the gelatinous capsule of the chorion (confirmed by fluorescence microscopy), which protected the embryos from eventual direct effects of the microplastics on the hatching and survival rates. Although there were no cardiotoxic effects nor morphometric alterations, there was a significant increase in abdominal edemas in the hatched embryos of the PE MPs group, which indicates that osmoregulation might have been affected by the attachment of the microplastics on the embryos' gelatinous capsule. This study presents the first evidence of developmental toxicity of environmental mixtures of microplastics and nanoparticles on amphibians and reinforces the need for more studies with other amphibian species, especially neotropical specimens that could present bigger sensibility. Our study also highlighted several features of the FETAX protocol as useful tools to evaluate the embryotoxicity of several pollutants on amphibians.

How hibernation in frogs drives brain and reproductive evolution in opposite directions.

Environmental seasonality can promote the evolution of larger brains through cognitive and behavioral flexibility but can also hamper it when temporary food shortage is buffered by stored energy. Multiple hypotheses linking brain evolution with resource acquisition and allocation have been proposed for warm-blooded organisms, but it remains unclear how these extend to cold-blooded taxa whose metabolism is tightly linked to ambient temperature. Here, we integrated these hypotheses across frogs and toads in the context of varying brumation (hibernation) durations and their environmental correlates. We showed that protracted brumation covaried negatively with brain size but positively with reproductive investment, likely in response to brumation-dependent changes in the socio-ecological context and associated selection on different tissues. Our results provide novel insights into resource allocation strategies and possible constraints in trait diversification, which may have important implications for the adaptability of species under sustained environmental change.

Convergence of gut microbiota in myrmecophagous amphibians.

The gut microbiome composition of terrestrial vertebrates is known to converge in response to common specialized dietary strategies, like leaf-eating (folivory) or ant- and termite-eating (myrmecophagy). To date, such convergence has been studied in mammals and birds, but has been neglected in amphibians. Here, we analysed 15 anuran species (frogs and toads) representing five Neotropical families and demonstrated the compositional convergence of the gut microbiomes of distantly related myrmecophagous species. Specifically, we found that the gut microbial communities of bufonids and microhylids, which have independently evolved myrmecophagy, were significantly more similar than expected based on their hosts' evolutionary divergence. Conversely, we found that gut microbiome composition was significantly associated with host evolutionary history in some cases. For instance, the microbiome composition of Xenohyla truncata, one of the few known amphibians that eat fruits, was not different from those of closely related tree frogs with an arthropod generalist diet. Bacterial taxa overrepresented in myrmecophagous species relative to other host families include Paludibacter, Treponema, and Rikenellaceae, suggesting diet-mediated selection and prey-to-predator transmission likely driving the observed compositional convergence. This study provides a basis for examining the roles of the gut microbiome in host tolerance and sequestration of toxic alkaloids from ants and termites.

Climate change in the Central Amazon and its impacts on frog populations.

Frog population declines have already been observed in the central Amazon even for common species that are considered not to be in danger of extinction. The Amazon is close to its limit of tolerated deforestation, and parts of the forest have already been modified by climate change, which raises questions about how the fauna in these areas would adapt to climate changes by the middle and the end of this century. In this study we used population density data on seven species of Amazonian frogs and analyzed the relationship between the activity of these species and temperature, precipitation, and relative humidity. We also used the least-squares method with logarithmic models to assess whether climate change projected by the Intergovernmental Panel on Climate Change (IPCC) would be an indicator of the population dynamics of these species. Our results suggest that even common species may be may experience population declines and extinction in the next decades due to climate changes.

Amphibian diversity across three adjacent ecosystems in Área de Conservación Guanacaste, Costa Rica.

Amphibians are the most threatened species-rich vertebrate group, with species extinctions and population declines occurring globally, even in protected and seemingly pristine habitats. These 'enigmatic declines' are generated by climate change and infectious diseases. However, the consequences of these declines are undocumented as no baseline ecological data exists for most affected areas. Like other neotropical countries, Costa Rica, including Área de Conservación Guanacaste (ACG) in north-western Costa Rica, experienced rapid amphibian population declines and apparent extinctions during the past three decades. To delineate amphibian diversity patterns within ACG, a large-scale comparison of multiple sites and habitats was conducted. Distance and time constrained visual encounter surveys characterised species richness at five sites-Murciélago (dry forest), Santa Rosa (dry forest), Maritza (mid-elevation dry-rain forest intersect), San Gerardo (rainforest) and Cacao (cloud forest). Furthermore, species-richness patterns for Cacao were compared with historic data from 1987-8, before amphibians declined in the area. Rainforests had the highest species richness, with triple the species of their dry forest counterparts. A decline of 45% (20 to 11 species) in amphibian species richness was encountered when comparing historic and contemporary data for Cacao. Conservation efforts sometimes focus on increasing the resilience of protected areas, by increasing their range of ecosystems. In this sense ACG is unique containing many tropical ecosystems compressed in a small geographic space, all protected and recognised as a UNESCO world heritage site. It thus provides an extraordinary platform to understand changes, past and present, and the resilience of tropical ecosystems and assemblages, or lack thereof, to climate change.

Tracking the Diversity and Chromosomal Distribution of the Olfactory Receptor Gene Repertoires of Three Anurans Species.

Olfaction is a crucial capability for most vertebrates and is realized through olfactory receptors in the nasal cavity. The enormous diversity of olfactory receptors has been created by gene duplication, following a birth-and-death model of evolution. The olfactory receptor genes of the amphibians have received relatively little attention up to now, although recent studies have increased the number of species for which data are available. This study analyzed the diversity and chromosomal distribution of the OR genes of three anuran species (Engystomops pustulosus, Bufo bufo and Hymenochirus boettgeri). The OR genes were identified through searches for homologies, and sequence filtering and alignment using bioinformatic tools and scripts. A high diversity of OR genes was found in all three species, ranging from 917 in B. bufo to 1194 in H. boettgeri, and a total of 2076 OR genes in E. pustulosus. Six OR groups were recognized using an evolutionary gene tree analysis. While E. pustulosus has one of the highest numbers of genes of the gamma group (which detect airborne odorants) yet recorded in an anuran, B. bufo presented the smallest number of pseudogene sequences ever identified, with no pseudogenes in either the beta or epsilon groups. Although H. boettgeri shares many morphological adaptations for an aquatic lifestyle with Xenopus, and presented a similar number of genes related to the detection of water-soluble odorants, it had comparatively far fewer genes related to the detection of airborne odorants. This study is the first to describe the complete OR repertoire of the three study species and represents an important contribution to the understanding of the evolution and function of the sense of smell in vertebrates.

Female treefrog preference for breeding sites matches offspring performance in the presence of two anuran competitors.

Preference-performance theory predicts that females should select breeding sites that maximize offspring performance. Amphibians have been a model system for investigating habitat selection, yet most studies have focused on habitat selection in response to predators and conspecifics. We investigated female oviposition site selection and larval performance in eastern gray treefrogs (Hyla versicolor) among pools with and without one of two ecologically distinct heterospecific larvae, the green frog (Rana clamitans) and the American bullfrog (Rana catesbeiana). Pools without heterospecifics were used on more nights and had more eggs deposited compared to pools with heterospecifics. In a competition experiment, treefrog larval performance matched this female preference: tadpoles developed faster and grew larger in the absence of either heterospecific. Although we hypothesized that bullfrogs would more strongly affect female treefrog preference and offspring performance because of previous work demonstrating that bullfrogs had stronger negative effects on other tadpoles, both heterospecifics elicited similar responses. The effects of heterospecifics on anuran breeding site selection are understudied and not well understood, and our results demonstrate that female selection of breeding sites is an adaptive behavior for offspring in the presence of heterospecific competitors.

Frog phylogeny: A time-calibrated, species-level tree based on hundreds of loci and 5,242 species.

Large-scale, time-calibrated phylogenies from supermatrix studies have become crucial for evolutionary and ecological studies in many groups of organisms. However, in frogs (anuran amphibians), there is a serious problem with existing supermatrix estimates. Specifically, these trees are based on a limited number of loci (15 or fewer), and the higher-level relationships estimated are discordant with recent phylogenomic estimates based on much larger numbers of loci. Here, we attempted to rectify this problem by generating an expanded supermatrix and combining this with data from phylogenomic studies. To assist in aligning ribosomal sequences for this supermatrix, we developed a new program (TaxonomyAlign) to help perform taxonomy-guided alignments. The new combined matrix contained 5,242 anuran species with data from 307 markers, but with 95% missing data overall. This dataset represented a 71% increase in species sampled relative to the previous largest supermatrix analysis of anurans (adding 2,175 species). Maximum-likelihood analyses generated a tree in which higher-level relationships (and estimated clade ages) were generally concordant with those from phylogenomic analyses but were more discordant with the previous largest supermatrix analysis. We found few obvious problems arising from the extensive missing data in most species. We also generated a set of 100 time-calibrated trees for use in comparative analyses. Overall, we provide an improved estimate of anuran phylogeny based on the largest number of combined taxa and markers to date. More broadly, we demonstrate the potential to combine phylogenomic and supermatrix analyses in other groups of organisms.

Geographic Variation in Organ Size in a Toad (Duttaphrynus melanostictus).

Adaptive evolution is the process by which organisms change their morphological, physiological and biochemical characteristics to adapt to different environments during long-term natural selection. Especially, researching variation in organ size can provide important insights into morphological adaptation in amphibians. In this study, we comparatively studied differences in organ sizes (heart, lungs, liver, gallbladder, kidneys, spleen, digestive tract, testes and brain) among five geographical populations of the Asian common toad Duttaphrynus melanostictus. Our results revealed significant variations in the size of these nine specific organs among the populations. Notably, we observed a significant positive correlation between the relative size of the testes and latitude and/or altitude. However, no correlation was found between the relative size of the heart and the length of the digestive tract with altitude across populations, respectively, contradicting Hesse's rule and the digestion theory. These findings suggest that our study does not provide substantial theoretical support for the adaptive evolution of organ size in this particular toad species, but rather contributes to the understanding of the evolution and adaptations of species' different environmental conditions. Further research is warranted to delve deeper into the factors influencing organ size in amphibian populations.

Comparative genomics reveals insights into anuran genome size evolution.

BACKGROUND: Amphibians, particularly anurans, display an enormous variation in genome size. Due to the unavailability of whole genome datasets in the past, the genomic elements and evolutionary causes of anuran genome size variation are poorly understood. To address this, we analyzed whole-genome sequences of 14 anuran species ranging in size from 1.1 to 6.8 Gb. By annotating multiple genomic elements, we investigated the genomic correlates of anuran genome size variation and further examined whether the genome size relates to habitat types. RESULTS: Our results showed that intron expansions or contraction and Transposable Elements (TEs) diversity do not contribute significantly to genome size variation. However, the recent accumulation of transposable elements (TEs) and the lack of deletion of ancient TEs primarily accounted for the evolution of anuran genome sizes. Our study showed that the abundance and density of simple repeat sequences positively correlate with genome size. Ancestral state reconstruction revealed that genome size exhibits a taxon-specific pattern of evolution, with families Bufonidae and Pipidae experiencing extreme genome expansion and contraction events, respectively. Our result showed no relationship between genome size and habitat types, although large genome-sized species are predominantly found in humid habitats. CONCLUSIONS: Overall, our study identified the genomic element and their evolutionary dynamics accounting for anuran genome size variation, thus paving a path to a greater understanding of the size evolution of the genome in amphibians.

Biogeography and the evolution of acoustic communication in the polyploid North American grey treefrog complex.

After polyploid species are formed, interactions between diploid and polyploid lineages may generate additional diversity in novel cytotypes and phenotypes. In anurans, mate choice by acoustic communication is the primary method by which individuals identify their own species and assess suitable mates. As such, the evolution of acoustic signals is an important mechanism for contributing to reproductive isolation and diversification in this group. Here, we estimate the biogeographical history of the North American grey treefrog complex, consisting of the diploid Hyla chrysoscelis and the tetraploid Hyla versicolor, focusing specifically on the geographical origin of whole genome duplication and the expansion of lineages out of glacial refugia. We then test for lineage-specific differences in mating signals by applying comparative methods to a large acoustic data set collected over 52 years that includes >1500 individual frogs. Along with describing the overall biogeographical history and call diversity, we found evidence that the geographical origin of H. versicolor and the formation of the midwestern polyploid lineage are both associated with glacial limits, and that the southwestern polyploid lineage is associated with a shift in acoustic phenotype relative to the diploid lineage with which they share a mitochondrial lineage. In H. chrysoscelis, we see that acoustic signals are largely split by Eastern and Western lineages, but that northward expansion along either side of the Appalachian Mountains is associated with further acoustic diversification. Overall, results of this study provide substantial clarity on the evolution of grey treefrogs as it relates to their biogeography and acoustic communication.

Effects of anthropogenic light on anuran calling site.

The natural environment can be negatively impacted by a variety of human activities, including the production of artificial light at night. Recent studies suggest that pollution from anthropogenic light alters animal behavior. Despite being highly nocturnal, little attention has been given to anurans and the effects artificial light at night has on their behavior. This study investigated whether artificial light influenced male call site selection in east Texas anurans. Ambient light levels were quantified at five sites that varied in urbanization and artificial light levels. Calling males were located and ambient light was then measured at the male's call location. Light levels at those call locations were compared to the general light environment as measured at random locations in the area. There was a consistent pattern where males at the brightest sites called from locations darker than the general light environment. However, male call locations at the brightest sites were generally brighter than those at the darker sites suggesting that, while male anurans avoid illuminated areas for calling, males in more urbanized populations may be unable to do so. As such, male anurans at sites with higher light pollution may experience a form of habitat loss where preferred darker habitat is not available.

Proteins from toad's parotoid macroglands: do they play a role in gland functioning and chemical defence?

BACKGROUND: Parotoid gland secretion of bufonid toads is a rich source of toxic molecules that are used against predators, parasites and pathogens. Bufadienolides and biogenic amines are the principal compounds responsible for toxicity of parotoid secretion. Many toxicological and pharmacological analyses of parotoid secretions have been performed, but little is known about the processes related to poison production and secretion. Therefore, our aim was to investigate protein content in parotoids of the common toad, Bufo bufo, to understand the processes that regulate synthesis and excretion of toxins as well as functioning of parotoid macroglands. RESULTS: Applying a proteomic approach we identified 162 proteins in the extract from toad's parotoids that were classified into 11 categories of biological functions. One-third (34.6%) of the identified molecules, including acyl-CoA-binding protein, actin, catalase, calmodulin, and enolases, were involved in cell metabolism. We found many proteins related to cell division and cell cycle regulation (12.0%; e.g. histone and tubulin), cell structure maintenance (8.4%; e.g. thymosin beta-4, tubulin), intra- and extracellular transport (8.4%), cell aging and apoptosis (7.3%; e.g. catalase and pyruvate kinase) as well as immune (7.0%; e.g. interleukin-24 and UV excision repair protein) and stress (6.3%; including heat shock proteins, peroxiredoxin-6 and superoxide dismutase) response. We also identified two proteins, phosphomevalonate kinase and isopentenyl-diphosphate delta-isomerase 1, that are involved in synthesis of cholesterol which is a precursor for bufadienolides biosynthesis. STRING protein-protein interaction network predicted for identified proteins showed that most proteins are related to metabolic processes, particularly glycolysis, stress response and DNA repair and replication. The results of GO enrichment and KEGG analyses are also consistent with these findings. CONCLUSION: This finding indicates that cholesterol may be synthesized in parotoids, and not only in the liver from which is then transferred through the bloodstream to the parotoid macroglands. Presence of proteins that regulate cell cycle, cell division, aging and apoptosis may indicate a high epithelial cell turnover in parotoids. Proteins protecting skin cells from DNA damage may help to minimize the harmful effects of UV radiation. Thus, our work extends our knowledge with new and important functions of parotoids, major glands involved in the bufonid chemical defence.

MULTISYSTEMIC EMPHYSEMA (GAS BUBBLE DISEASE)-ASSOCIATED ACUTE MASS MORTALITY IN A FREE-RANGING POPULATION OF COMMON FROG (RANA TEMPORARIA) IN SWITZERLAND.

In April 2020, nearly 5,000 free-ranging common frogs (Rana temporaria) were found dead on the surface of the water in a retention pond in the Swiss Alps. Macroscopic and microscopic lesions revealed multisystem emphysema, affecting multiple organs. The most severe lesions were seen in the skin, eyes, and blood vessels of internal organs and were secondary to the sudden massive distension of the skin and other affected organs. All frogs had similar lesions consistent with those described associated with gas bubble disease. No obvious pre-existing conditions potentially priming the occurrence of the observed lesions could be detected. All the examined frogs were negative by PCR for Batrachochytrium dendrobatidis, Ranavirus and Ranid Herpesvirus 3 (now Batravirus ranidallo 3). The proposed etiology is considered to be an undetermined physical event, leading to an abrupt change in the molecular or physical characteristics of the water (namely pressure and oxygen or other gas supersaturation), resulting in the occurrence of the observed lesions in the frogs. No obvious pumping system malfunction was recorded in the Mägisalp ponds before the mass mortality, but a sudden and temporary undetected change in the water flow, which then quickly rebalanced, cannot be excluded. Other hypotheses include weather conditions, such as lightning strikes in the water, or a device detonating in the water.