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As a result of the research, 109 individuals of two amphibian species: the common frog (Rana temporaria) and the common toad (Bufo bufo) were selected. These two species are the most numerous among other species of amphibians within the studied territory, therefore, they can be considered typical species in the anthropogenically transformed habitats of the Ukrainian Roztochchia. During the analysis of the collected material, Ivlev’s selectivity index was used to determine the selective attitude of the studied amphibian species to the number of consumed objects. Comparing data obtained from Barber’s traps and material extracted from the stomachs of amphibians, it was concluded that depending on the habitat type, both the common frog (Rana temporaria) and the common toad (Bufo bufo) show a certain feeding selectivity. The common toad consumes mostly the representatives of Annelida (15%), Arachnida (15%) and Coleoptera (18%), what makes up 48% of all consumed objects in total. The common frog, under the conditions of anthropogenically transformed environment, prefers Mollusca (15%), Myriapoda (20%) and Hemiptera (20%) mostly, what is 55% in total. In half of the stomachs we examined for, both amphibian species, plant residues were found, specifically parts of leaves and branches. Additionally, in individuals of the Bufo bufo species, stones were discovered in the stomach. Most likely, these items ended up there accidentally during the capture of primary food objects. Species of tailless amphibians are highly vulnerable to the negative impact of various anthropogenic factors. Deforestation, water bodies drainage, and global or local environmental pollution can significantly affect diverse ecosystems. This, in turn, may lead to the disappearance of segments of the dietary spectrum for amphibians. Monitoring the trophic interactions of this animal class is crucial, given the importance of amphibians as vital links in food chains. This factor underscores the need for periodic trophic monitoring, as amphibians play a pivotal role in sustaining ecological balance within their vulnerable populations.

The paper presents data on the identification of the helminth fauna structure of Bufo bufo Linnaeus, 1758 and Bufotes viridis Laurenti, 1768 species, ecaudata amphibian from Bufonidae families, and the determination of its role as bioindicators and as vectors for parasitic agents specific to animals. As result of helminthological investigations during 2013-2023 years, in Bufonidae species 19 helminths species was established. The helminthological research carried out on the species Bufo bufo highlighted the presence of 19 species of helminths, of which the predominant parasitic agents are trematodes which make up 47.4%, nematodes with 31.5%, acanthocephals with 15.8% and monogeneans with 5.3%. In Bufotes viridis species, the presence of 18 species of helminths was established, of which helminth species from the trematode class represent the predominant group with 44.4%, nematodes with 38.9%, acanthocephales with 11.1% and monogeneans with 5.6%. The helminthological researches were carried out depending on the age of the host, the type of habitat and the area. The bioindicator significance of the established parasite species was presented for an ecological assessment of the studied area. [ABSTRACT FROM AUTHOR]

Abstract 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.

Input of different spectral types of photoreceptors in amphibian colour vision has been assessed by studying the behaviour of male toads Bufo bufo and B. gargarizans in a laboratory. This method is based on the males' innate reaction of approaching and clasping any visual object during the breeding season. A pair of colour stimuli made of paper or displayed on a LCD monitor has been selected to match the toad's RGB colour space so that they differ by excitation of only a single spectral class of retinal photoreceptors. With pairwise presentation, the males are found to prefer a more 'bluish', or a less 'reddish', or a less 'greenish' stimulus from each pair. A microspectrophotometry of isolated rod and cone photoreceptors and an observation of behavioural responses in toads have revealed a trichromatic mesopic visual system based on a positive input from blue-sensitive photoreceptors and negative inputs from red-sensitive cones and green-sensitive rods. It is supposed that the observed colour preference and involvement of different chromatic mechanisms determine social interactions of anurans during mate choice. [ABSTRACT FROM AUTHOR]

A comparative study of the morphofunctional parameters of testicular (non-activated) and urinal (activated) spermatozoa of the common toad Bufo bufo (L., 1758) before and after cryopreservation was carried out. Cryopreservation was performed by a slow method. Sucrose (10%) and dimethylformamide (12%) were used as cryoprotectants. It was shown that the pool of testicular spermatozoa contains significantly more cells with abnormal morphology both before and after cryopreservation. The osmoticity of the base medium for the cryoprotective solution (Ringer's solution for amphibians – 230 mOsm or urinal plasma – 45 mOsm) had little effect on the survival of testicular and urinal spermatozoa during cryopreservation. Comparison of the morphological and functional characteristics of spermatozoa before and after freezing-thawing indicates that the cryoresistances of testicular and urinal spermatozoa differ slightly. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Throughout colonial Spanish America, the missionaries recreated a moralized bestiary drawn from medieval referents and applied to the fauna of the New World. This was the case of the cane toad, kururu (Rhinella diptycha), which was assimilated with the European common toad (Bufo bufo) in the Jesuit missions of Paraguay. In this context, it was used to speed up Lent confessions, embody the Christian concept of lust, and counteract the influence of shamans. This exemplary use of the toad was applied throughout the continent and triggered a paradoxical revival of shamanism, as demonstrated by sources in Guaraní, Nahuatl, and Quechua studied in a long-term perspective. [ABSTRACT FROM AUTHOR]

The helminths of fingerlings of the gray toad Bufo bufo (Linnaeus, 1758) have been studied in some specially protected natural areas (protected areas (PAs)) of the Urals. Only four species of helminths belonging to a Nematoda type are found. The nematodes Oswaldocruzia filiformis and Rhabdias bufonis are common for the studied populations of amphibians. The species composition of geohelminths does not differ in fingerlings or sexually mature amphibians; the infection in adult toads is higher than in fingerlings. The analysis of helminth fauna in the gray toad demonstrated that the nematodes Oswaldocruzia filiformis and Rhabdias bufonis dominate in Bufo bufo in most regions of Russia. The richest species composition of trematodes is noted in the gray toads of Mordovia. [ABSTRACT FROM AUTHOR]

We examined the cranial morphology and cranial kinesis of the common toads Bufo bufo and B. spinosus with micro-computed tomography and geometric morphometrics and compared the results with published data for related species in a phylogenetic context. The species significantly diverge in skull shape. The skull of B. spinosus is shorter and higher, with a ventral arm of the squamosal bone and the jaw articulation point positioned perpendicular to the braincase, in comparison with a more lateral position in B. bufo. In either species, females have a shorter snout and a higher and wider skull at the jaw articulation point that is positioned more posteriorly, in comparison with conspecific males. High variation in the amount of bone ossification was recorded in both species, ranging from scarcely ossified and loosely connected bones to highly ossified and firmly connected bones. We also found that skull shape and inferred kinetic properties of the skull are highly variable across the Bufonini tribe. However, sample sizes are mostly small and intraspecific variation is high, which might compromise the analyses. Overall, the results suggest that developmental plasticity produces high variation in ossification and cranial kinesis, affecting individuals' feeding performances. At the population level, this variation supports an efficient exploitation of the habitat and may promote morphological adaptation in a changing environment. [ABSTRACT FROM AUTHOR]

The gut microbiome is important for host health and can be influenced by environmental and hormonal changes. We studied the interactions between anthropogenic land use, glucocorticoid hormones, and gut bacterial communities in common toads (Bufo bufo). We sampled tadpoles from ponds of three habitat types (natural, agricultural, and urban ponds), examined gut microbiome composition using amplicon sequencing of the 16S rRNA gene, and measured the associated stress physiology using water-borne hormones. Tadpoles from different habitat types significantly differed in bacterial composition. However, bacterial richness, Shannon diversity, and Firmicutes to Bacteroidota ratio did not vary with habitat type. In contrast with other studies, we found a positive correlation between baseline corticosterone release rate and bacterial diversity. Stress response and negative feedback were not significantly correlated with bacterial diversity. These results suggest that, despite alterations in the composition of intestinal bacterial communities due to land-use change, common toad tadpoles in anthropogenic habitats may maintain their physiological health in terms of the "gut-brain axis". [ABSTRACT FROM AUTHOR]

Background: A growing number of reports indicate that amphibian skin secretions may have a remarkable medical importance; however, the effects of the components of some dermal secretions on blood platelets and hemostasis are inadequately recognized. Since our previous studies demonstrated that the general Bufo bufo skin secretions induced platelet aggregation in platelet-rich plasma, this work was designed to study the effects of the components of some fractions on platelet functions to comprehend its possible mechanism of action as platelet modulators. Methods: Chromatographic separation of B. bufo general skin secretions was carried out using size exclusion chromatography. Rabbit platelets were purified by column chromatography on Sepharose 4B. Various aspects of platelet function such as activation, aggregation, and adhesion were evaluated. Results: One fraction, out of 7, dose-dependently induced aggregation of isolated platelets and was used in further experiments. The studied fraction was shown to induce platelet adhesion onto fibrinogen-coated surface. Furthermore, the results demonstrated the effects of the fraction on some processes that involved in platelets activation: The fraction components facilitated (Ca2+) i mobilization and attenuated platelets Akt phosphorylation, but had no effect on platelet serotonin secretion. Membrane integrity was determined using lactate dehydrogenase (LDH) assay. No increased LDH release was recorded that means no platelet damage, which could lead to misinterpretation of the data, occurred. Conclusion: The results suggest that components of the B. bufo skin secretions may be a promising source of natural compounds which can modulate platelet functions. [ABSTRACT FROM AUTHOR]

Introduction: Erythrocytes are highly specialized and the most abundant cell type in vertebrates’ blood. Their primary function is the transportation of oxygen to tissues of the body via hemoglobin. Methods: The study was aimed at evaluating erythrocyte morphology in Clarias gariepinus (C. gariepinus), Bufo bufo (B. bufo), and Agama agama (A. agama) using different stains; Also, morphometric analysis of erythrocytes was evaluated in stains with the best affinity. The blood samples of C. gariepinus, B. bufo, and A. agama were collected using a syringe. Smears from each animal were made on glass slides and stained with Giemsa, Hematoxylin and eosin (H&E), and methylene blue. Erythrocytes diameter was measured using a standardized ocular micrometer. The area and volume of the erythrocytes were calculated. Results: Erythrocyte’s shapes range from spherical in C. gariepinus to oval in A. agama. Giemsa demonstrated C. gariepinus and B. bufo erythrocytes better than H&E and methylene blue stains, while H&E demonstrated erythrocytes of A. agama better than Giemsa and methylene blue. Erythrocyte’s diameter, area, and volume of B. bufo were significantly higher (P

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;20:2218-2230. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC)., (© 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).)

Associational effects such as "associational resistance" or "aposematic commensalism" may facilitate the subsequent evolution of mimicry. However, such effects are usually expected to be contingent on a spatiotemporal co-occurrence of defended and undefended species. Associational resistance may emerge in communities of larval anurans where tadpoles of different species share a generalized morphology but vary in chemical defences. In Europe, the marsh frog Pelophylax ridibundus reproduces in fish-containing habitats that earlier in the season can be used by the common toad Bufo bufo, a species with chemically defended larvae. In a mesocosm experiment, we investigated if the associational protection against predation provided by the defended species could be strong enough to last even after the defended prey metamorphose and leave the system. We expected that the higher abundances (both absolute and relative) of chemically defended B. bufo in the tadpole assemblage exposed to predation by the common carp Cyprinus carpio prior to the occurrence of P. ridibundus tadpoles would be related to the increased later survival of P. ridibundus in the presence of the same fish. The tadpoles of P. ridibundus were highly vulnerable to predation during a 2-week cohabitation with fish. We found no relationship between the survival of P. ridibundus and the absolute density or relative proportion of B. bufo. Although associational effects may be ecologically relevant in aquatic animal communities, they apparently require the co-presence of the defended prey in fish-tadpole interactions. [ABSTRACT FROM AUTHOR]

Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, an infectious disease of amphibians, which has contributed to population declines in hundreds of species worldwide. Common toads (Bufo bufo) exhibit low resistance and relatively high tolerance to Bd infection, which may partly be attributable to bufadienolide toxins secreted in their granular skin glands. Bufadienolides are known to provide an effective defense against several pathogens, parasites, and predators. The toxin production of bufonids is a plastic trait, inducible by several environmental factors. Here, we experimentally infected juvenile common toads with Bd and investigated if the toadlets could clear the infection over time, whether the infection induced bufadienolide production, and whether the infection caused decreased body mass. We found that prevalence remained 100% throughout the entire experimental period, but infection intensity did not increase and it was significantly lower on day 30 than on day 20. At the same time, compared to controls, infected toadlets produced lesser amounts of bufadienolides and their body mass was also lower. These results suggest that although young toadlets may not be able to clear Bd infection on their own, they may be able to keep infection intensities under control. Nonetheless, even if toadlets do not succumb to the disease, the costs of chronic infection may still compromise their fitness. [ABSTRACT FROM AUTHOR]

While both innate and adaptive immune system mechanisms have been implicated in resistance against the chytrid fungus Batrachochytrium dendrobatidis (Bd), studies on the role of specific MHC haplotypes on Bd infection are rare. Here, we studied variation in MHC Class IIB loci in the common toad Bufo bufo along a latitudinal gradient across Sweden. In general, Swedish toad populations had few MHC Class IIB haplotypes and MHC diversity declined from south (13 haplotypes) to the north (four haplotypes). The low diversity may compromise the ability of northern populations to fight emerging disease, such as Bd. In a laboratory experiment, we infected newly metamorphosed toads with two strains of the Global Pandemic Lineage of the fungus (Bd-GPL) and compared survival with sham controls. Bd-infected toads had lower survival compared to controls. Moreover, survival was dependent on the Bd-strain and northern toads had lower Bd-mediated survival than southern individuals. MHC diversity was lower in northern toads. All northern experimental animals were monomorphic for a single MHC haplotype, whereas we found seven different haplotypes in southern experimental animals. In southern toads, survival was dependent on both Bd-strain and MHC haplotype suggesting differential infection dynamics depending on both Bd-strain and host immune system characteristics.

The putatively positive association between host genetic diversity and the ability to defend against pathogens has long attracted the attention of evolutionary biologists. Chytridiomycosis, a disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has emerged in recent decades as a cause of dramatic declines and extinctions across the amphibian clade. Bd susceptibility can vary widely across populations of the same species, but the relationship between standing genetic diversity and susceptibility has remained notably underexplored so far. Here, we focus on a putatively Bd‐naive system of two mainland and two island populations of the common toad (Bufo bufo) at the edge of the species' range and use controlled infection experiments and dd‐RAD sequencing of >10 000 SNPs across 95 individuals to characterize the role of host population identity, genetic variation and individual body mass in mediating host response to the pathogen. We found strong genetic differentiation between populations and marked variation in their susceptibility to Bd. This variation was not, however, governed by isolation‐mediated genetic erosion, and individual heterozygosity was even found to be negatively correlated with survival. Individual survival during infection experiments was strongly positively related to body mass, which itself was unrelated to population of origin or heterozygosity. Our findings underscore the general importance of context‐dependency when assessing the role of host genetic variation for the ability of defence against pathogens. [ABSTRACT FROM AUTHOR]

Introduction: Erythrocytes are highly specialized and the most abundant cell type in vertebrates' blood. Their primary function is the transportation of oxygen to tissues of the body via hemoglobin. Methods: The study was aimed at evaluating erythrocyte morphology in Clarias gariepinus (C. gariepinus), Bufo bufo (B. bufo), and Agama agama (A. agama) using different stains; Also, morphometric analysis of erythrocytes was evaluated in stains with the best affinity. The blood samples of C. gariepinus, B. bufo, and A. agama were collected using a syringe. Smears from each animal were made on glass slides and stained with Giemsa, Hematoxylin and eosin (H&E), and methylene blue. Erythrocytes diameter was measured using a standardized ocular micrometer. The area and volume of the erythrocytes were calculated. Results: Erythrocyte's shapes range from spherical in C. gariepinus to oval in A. agama. Giemsa demonstrated C. gariepinus and B. bufo erythrocytes better than H&E and methylene blue stains, while H&E demonstrated erythrocytes of A. agama better than Giemsa and methylene blue. Erythrocyte's diameter, area, and volume of B. bufo were significantly higher (P<0.001) when compared with C. gariepinus and A. agama. Conclusion: Erythrocyte's shape, size, area, and volume vary in different species and could be used to study evolutionary trends. The variation in erythrocytes size is associated with chromosome number and genome size. [ABSTRACT FROM AUTHOR]

Abstract The growing interest in the lability of sex determination in non‐model vertebrates such as amphibians and fishes has revealed high rates of sex chromosome turnovers among closely related species of the same clade. Can such lineages hybridize and admix with different sex‐determining systems, or could the changes have precipitated their speciation? We addressed these questions in incipient species of toads (Bufonidae), where we identified a heterogametic transition and characterized their hybrid zone with genome‐wide markers (RADseq). Adult and sibship data confirmed that the common toad B. bufo is female heterogametic (ZW), while its sister species the spined toad B. spinosus is male heterogametic (XY). Analysis of a fine scale transect across their parapatric ranges in southeastern France unveiled a narrow tension zone (∼10 km), with asymmetric mitochondrial and nuclear admixture over hundreds of kilometers southward and northward, respectively. The geographic extent of introgression is consistent with an expansion of B. spinosus across B. bufo’s former ranges in Mediterranean France, as also suggested by species distribution models. However, widespread cyto‐nuclear discordances (B. spinosus backrosses carrying B. bufo mtDNA) run against predictions from the dominance effects of Haldane's rule, perhaps because Y and W heterogametologs are not degenerated. Common and spined toads can thus successfully cross‐breed despite fundamental differences in their sex determination mechanisms, but remain partially separated by reproductive barriers. Whether and how the interactions of their XY and ZW genes contribute to these barriers shall provide novel insights on the debated role of labile sex chromosomes in speciation.

Many features of the urban environment, such as a warmer microclimate, can positively affect populations of birds and mammals inhabiting cities, while at the same time may affect amphibians negatively. Also, amphibians from the temperate zone need both water bodies for reproduction and terrestrial habitats for foraging and hibernation, so changes in only one of these habitats may affect them negatively, and strong declines in many species are being observed. This study was conducted on two species, reacting differently to urbanization pressure – the common toad Bufo bufo and green toad Bufotes viridis. In the 1992–1994 breeding seasons, the occurrence of spawning toads in urban water bodies was established, and such a survey was repeated 15 years later (2007–2009). Over a shorter period, the breeding phenology, as well as the condition of breeding individuals, was studied. We found that after 15 years, a significant loss of the breeding sites of the two toad species took place, and the rate of their extinction was similar (39–47%), but green toad breeding ponds were located closer to the city center than before (2.4 vs 4.6 km). In both periods, the common toad spawned in water bodies located farther from the center (ca 7.2–8.7 km). The breeding phenology of both species was related to the minimal decade temperatures of April, and the green toad started to breed later than the common toad by 9–23 days. The condition of toad individuals differed between species, and after a colder winter, common toads were in better condition than green toads.

Dried toad skin (TS) and toad venom (TV) are the dried skin of the Bufo bufo gargarizans Cantor and the Bufo melanostictus Schneider , which remove the internal organs and the white secretions of the skin and retroauricular glands. Since 2005, cinobufacini preparations have been approved by the State Food and Drug Administration for use as adjuvant therapies in the treatment of various advanced cancers. Meanwhile, bufalenolides has been identified as the main component of TS/TV, exhibiting antitumor activity, inducing apoptosis of cancer cells and inhibiting cancer cell proliferation or metastasis through a variety of signaling pathways. However, clinical agents frequently face limitations such as inherent toxicity at high concentrations and insufficient tumor targeting. Additionally, the development and utilization of these active ingredients are hindered by poor water solubility, low bioavailability, and rapid clearance from the bloodstream. To address these challenges, the design of a targeted drug delivery system (TDDS) aims to enhance drug bioavailability, improve targeting within the body, increase drug efficacy, and reduce adverse reactions. This article reviews the TDDS for TS/TV, and their active components, including passive, active, and stimuli-responsive TDDS, to provide a reference for advancing their clinical development and use., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Zhang et al.)

The gut microbiome is important for host health and can be influenced by environmental and hormonal changes. We studied the interactions between anthropogenic land use, glucocorticoid hormones, and gut bacterial communities in common toads (Bufo bufo). We sampled tadpoles from ponds of three habitat types (natural, agricultural, and urban ponds), examined gut microbiome composition using amplicon sequencing of the 16S rRNA gene, and measured the associated stress physiology using water-borne hormones. Tadpoles from different habitat types significantly differed in bacterial composition. However, bacterial richness, Shannon diversity, and Firmicutes to Bacteroidota ratio did not vary with habitat type. In contrast with other studies, we found a positive correlation between baseline corticosterone release rate and bacterial diversity. Stress response and negative feedback were not significantly correlated with bacterial diversity. These results suggest that, despite alterations in the composition of intestinal bacterial communities due to land-use change, common toad tadpoles in anthropogenic habitats may maintain their physiological health in terms of the “gut-brain axis”.

Accumulating evidence shows that the body size of organisms in many taxa is declining in response to global warming. We investigated the potential effect of rising temperatures on the body size of a common toad (Bufo bufo L.) population in southern England between 1983 and 2020. The body length (SVL; in millimetres) and body mass (in grams) of 15 550 males and 4004 females arriving at their breeding pond for the first time were recorded. Toad body condition was estimated using a scaled body mass index (SMI). Over the study period, the mean annual temperature increased by ~1.3 °C, whereas the mean SVL and SMI of both sexes declined. Multiple regression analysis showed that female and male SMI were negatively correlated with increases in mean summer temperatures, with females also being impacted minimally by mean winter rainfall. Mean body size (SVL) was positively correlated with toad abundance over the period between emergence as toadlets and arrival at the pond to breed for the first time. Common toads exhibited phenotypic plasticity in response to warming environmental conditions, resulting in a reduction in SMI and subsequent reductions in SVL and overall egg production, which occurred over a period of 10 years. [ABSTRACT FROM AUTHOR]

The potential impact of pairing on male survivorship and future breeding success in common toads was investigated between 1993 and 2014. A total of 8132 males arriving at a breeding pond in southern England were measured, weighed, individually marked and their breeding history recorded. The body condition (BC) of all males was estimated using (a) residuals from an ordinary least squares regression analysis of log10 body mass against log10 body snout-vent length (SVL; ordinary least squares) and (b) a scaled mass index (SMI), standardized for SVL. Each year each male was allocated to one of four categorical groups: (1) Paired, (2) Unpaired, and whether or not it returned to breed the following year: (3) Return, (4) No Return. The BC of both paired and unpaired males that subsequently returned was significantly higher than that of those that failed to return. Male pairing success was highest in the youngest males and lowest in the oldest. The overall cost for male common toads that successfully paired was a significantly decreased BC and survival rate. First time breeding males had a 21.2% chance of surviving and returning to breed the following year, increasing to 28.3–37.0% for those returning for up to five consecutive years. [ABSTRACT FROM AUTHOR]

Processes of island colonisation have long been of interest to biologists. Colonisation events themselves are rarely observed, but the processes involved may be inferred using genetic approaches. We investigated possible means of island colonisation by common toads (Bufo bufo) in western Scotland (the Isle of Skye and five neighbouring small islands), using evidence derived from nuclear microsatellites and mitochondrial (mt) DNA. Levels of microsatellite allelic richness for populations on Skye were high and comparable to adjacent mainland populations, but lower for populations on small islands. Pairwise measures of genetic distances between populations and a clustering algorithm were both suggestive of frequent gene flow between Skye and the mainland. For small islands the levels of genetic differentiation were higher, implying stronger isolation and no evidence for inbreeding. The distribution of mtDNA haplotypes broadly mirrored the genetic structure revealed by microsatellites. Reconciled with existing palaeoclimatological evidence, since the last glaciation, our findings rule out the possibility that the B. bufo populations stem from glacial refugia, or that recent anthropogenic transfer of toads is responsible for their current distribution. The most parsimonious explanation of our data is that the studied inshore islands have been repeatedly colonised via rafting from the mainland or neighbouring islands. This may give us insights into the processes likely to take place when ice sheets retreat poleward as a result of climate change. It also has implications for the colonisation of both native and invasive non-native species, and hence the biosecurity of island refugia. [ABSTRACT FROM AUTHOR]

We present a genome assembly from an individual male Bufo bufo (the common toad; Chordata; Amphibia; Anura; Bufonidae). The genome sequence is 5.04 gigabases in span. The majority of the assembly (99.1%) is scaffolded into 11 chromosomal pseudomolecules. Gene annotation of this assembly by the NCBI Eukaryotic Genome Annotation Pipeline has identified 21,517 protein coding genes.

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

Objective of research: Characteristics of the species composition of parasites in common toad (Bufo bufo Linnaeus, 1758) inhabiting around Visimsky State Nature Biosphere Reserve. Materials and methods: 29 individuals of B. bufo were studied by the method of total helminthological autopsy. Catching of toads was carried out in the upstream flow of the Sulem River in the buffer zone of the Visimskiy Reserve during the reproduction period. Results and discussion: The species composition of parasites of B. bufo inhabiting Visimsky Biosphere Reserve (the upper Sulem river) has been studied for the first time. 5 parasite species were detected: Nematoda - Oswaldocruzia filiformis (Goeze, 1782), Neoraillietnema praeputiale (Skrjabin, 1916), Cosmocerca ornata (Dujardin, 1845), Rhabdias bufonis (Schrank, 1788) and 1 protozoa Opalinatea - Opalina ranarum (Purkinje et Valentin, 1835). The extensity of infection (EI) of common toads with helminths was 100 %, the index of abundance 47 %; protozoa infections 4 % and 0,45, respectively. Two nematode species O. filiformis (EI 100 %, index of abundance 27,8%) and R. bufonis (EI 89,7 %, index of abundance 13,3) dominated. In B. bufo selected for investigation no trematodes were found likely due to the «marriage post».

Genetic diversity feeds the evolutionary process and allows populations to adapt to environmental changes. However, we still lack a thorough understanding of why hotspots of genetic diversity are so 'hot'. Here, we analysed the relative contribution of bioclimatic stability and genetic admixture between divergent lineages in shaping spatial patterns of genetic diversity in the common toad Bufo bufo along the Italian peninsula. We combined population genetic, phylogeographic and species distribution modelling (SDM) approaches to map ancestral areas, glacial refugia, and secondary contact zones. We consistently identified three phylogeographic lineages, distributed in northern, central and southern Italy. These lineages expanded from their ancestral areas and established secondary contact zones, before the last interglacial. SDM identified widespread glacial refugia in peninsular Italy, sometimes located under the present-day sea-level. Generalized linear models indicated genetic admixture as the only significant predictor of the levels of population genetic diversity. Our results show that glacial refugia contributed to preserving both levels and patterns of genetic diversity across glacial-interglacial cycles, but not to their formation, and highlight a general principle emerging in Mediterranean species: higher levels of genetic diversity mark populations with substantial contributions from multiple genetic lineages, irrespective of the location of glacial refugia. [ABSTRACT FROM AUTHOR]

We studied the effect of antibiotic gentamicin at concentrations of 0.05, 0.1, 0.2, 0.4, and 1 mg/ml on the maintenance of sperm motility of the common toad Bufo bufo during cold storage of spermic urine samples at 4°C. Parameters of sperm motility during storage of samples with gentamicin at concentrations of 0.05-0.4 mg/ml did not differ significantly, but were higher (p<0.0001) than in the control (storage without antibiotic). Gentamicin at a concentration of 1 mg/ml had a negative effect on sperm motility. After 2 weeks of storage of toad spermic urine samples with gentamicin, the largest number of sperm was preserved when using antibiotic at a concentration of 0.4 mg/ml., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Agrochemicals cause diverse effects on aquatic communities, and amphibian species are particularly threatened due the high susceptibility to contamination. Present study evaluates the toxicity of a widely used fungicide tebuconazole (Trigal®) by the assessment of mortality and developmental alterations at acute, subchronic, and chronic exposure during the embryo-larval development of the South American toad Rhinella arenarum. Also, the sensitivity of the different embryonic stages was evaluated with 24-h pulse exposure treatments. The results demonstrated that larvae were more sensitive than embryos at acute exposure (LC50-24 and 96 h = 74.62, 31.92 mg/L and 24.27, 16.81 mg/L for embryos and larvae, respectively). Nevertheless, embryos toxicity increased significantly achieving a sensitivity very similar to larvae at chronic exposure (LC50-168 and 504 h = 13.31, 4.35 mg/L and 14.47, 6.83 mg/L for embryos and larvae, respectively). Embryos exhibited several sublethal effects from 5 mg/L at 96 h onwards, such as delayed development, reduce body size, edemas, tail/axial flexures, weakness, and absence of movements. The teratogenic index at 96 h was 10.13, indicating the severe teratogenic potential of the fungicide. 24-h pulse exposure treatments showed an increased sensitivity in intermediate stages as S.11, S.18, S20, and S.23 (NOEC-96 h = 100, 200, 75, and 20 mg/L, respectively), while stage S.25 was the most sensitive to the fungicide (NOEC-96 h = 5 mg/L). About metamorphic process, tebuconazole caused an acceleration of metamorphosis at the lowest concentration (0.001 mg/L), but also an increase in mortality and in addition, significant differences in the weight in all treatments. The results obtained throughout this work indicate that tebuconazole cause several adverse effects in Rhinella arenarum embryo-larval development., (© 2023 Wiley Periodicals LLC.)

Many features of the urban environment, such as a warmer microclimate, can positively affect populations of birds and mammals inhabiting cities, while at the same time may affect amphibians negatively. Also, amphibians from the temperate zone need both water bodies for reproduction and terrestrial habitats for foraging and hibernation, so changes in only one of these habitats may affect them negatively, and strong declines in many species are being observed. This study was conducted on two species, reacting differently to urbanization pressure – the common toad Bufo bufo and green toad Bufotes viridis. In the 1992–1994 breeding seasons, the occurrence of spawning toads in urban water bodies was established, and such a survey was repeated 15 years later (2007–2009). Over a shorter period, the breeding phenology, as well as the condition of breeding individuals, was studied. We found that after 15 years, a significant loss of the breeding sites of the two toad species took place, and the rate of their extinction was similar (39–47%), but green toad breeding ponds were located closer to the city center than before (2.4 vs 4.6 km). In both periods, the common toad spawned in water bodies located farther from the center (ca 7.2–8.7 km). The breeding phenology of both species was related to the minimal decade temperatures of April, and the green toad started to breed later than the common toad by 9–23 days. The condition of toad individuals differed between species, and after a colder winter, common toads were in better condition than green toads. [ABSTRACT FROM AUTHOR]

Amphibians are an important but vulnerable component of biodiversity. Climatic changes and anthropogenic transformation of the environment can lead to changes in spawning times and habitat boundaries, causing adverse genetic processes in populations. In the present study, we assess genetic variation and differentiation among 184 individuals of four amphibian species (Rana arvalis, Rana amurensis, Rana temporaria, Bufo bufo) from seven localities of the south of Western Siberia and the Urals, Russia. To investigate the genetic diversity of these species, we used six primers for inter simple sequences repeat (ISSR) markers. Nei's gene diversity (h) varied from 0.169 to 0.311 in the local populations of amphibians; the diversity was the smallest in the common toad B. bufo and the highest in the Siberian wood frog R. amurensis. Populations of B. bufo and the moor frog R. arvalis were highly differentiated (mean multilocus GST = 0.249 and 0.268, respectively). Nei's original measures of genetic identity (I) and genetic distance (D) among the toad populations were comparable with these indexes among the studied brown frog populations. These results indicate that B. bufo and R. arvalis have a well-defined population structure with restricted gene flow between populations. We also identified a high level of genetic diversity among eggs of R. arvalis not observed in adults. Our results provide genetic evidence that all the studied species have high adaptive potential and genetic structure typical for amphibian populations. The presented data are intended to fill the gap in studying the genetic structure of the amphibian populations of the south of Western Siberia and the Urals. The data on different levels of genetic variability in amphibian populations from protected areas show their different value for conservation management. The presence of genetically impoverished populations requires monitoring of genetic diversity of amphibians. These data will be useful for conservation concerns, especially for developing appropriate management strategies.

Using micro-CT and 3D landmark-based geometric morphometrics, I investigated postmetamorphic shape variation in the skull of Bufo bufo and Bufo spinosus, two widespread European toad species with small phenotypic differences. Two ontogenetic series were compared, for a total of 58 individuals. They exhibited similar allometric growth patterns, characterised by cranial widening with relative shortening and dorsoventral compression. However, some interspecific shape divergence was observed, particularly among adults: a relatively shorter skull and a more dorsally extended snout distinguished B. spinosus from B. bufo. This disparity, which gives further support to species separation, can probably be ascribed to changes in the allometric trajectories, and seen in light of the evolutionary history of the two lineages.

The growing interest in the lability of sex determination in non‐model vertebrates such as amphibians and fishes has revealed high rates of sex chromosome turnovers among closely related species of the same clade. Can such lineages hybridize and admix with different sex‐determining systems, or could the changes have precipitated their speciation? We addressed these questions in incipient species of toads (Bufonidae), where we identified a heterogametic transition and characterized their hybrid zone with genome‐wide markers (RADseq). Adult and sibship data confirmed that the common toad B. bufo is female heterogametic (ZW), while its sister species the spined toad B. spinosus is male heterogametic (XY). Analysis of a fine scale transect across their parapatric ranges in southeastern France unveiled a narrow tension zone (∼10 km), with asymmetric mitochondrial and nuclear admixture over hundreds of kilometers southward and northward, respectively. The geographic extent of introgression is consistent with an expansion of B. spinosus across B. bufo's former ranges in Mediterranean France, as also suggested by species distribution models. However, widespread cyto‐nuclear discordances (B. spinosus backrosses carrying B. bufo mtDNA) run against predictions from the dominance effects of Haldane's rule, perhaps because Y and W heterogametologs are not degenerated. Common and spined toads can thus successfully cross‐breed despite fundamental differences in their sex determination mechanisms, but remain partially separated by reproductive barriers. Whether and how the interactions of their XY and ZW genes contribute to these barriers shall provide novel insights on the debated role of labile sex chromosomes in speciation. [ABSTRACT FROM AUTHOR]

The interplay between intrinsic (development, physiology, behavior) and extrinsic (landscape features, climate) factors determines the outcome of admixture processes in hybrid zones, in a continuum from complete genetic merger to full reproductive isolation. Here we assess the role of environmental correlates in shaping admixture patterns in the long hybrid zone formed by the toads Bufo bufo and B. spinosus in western Europe. We used species-specific diagnostic SNP markers to genotype 6584 individuals from 514 localities to describe the contact zone and tested for association with topographic, bioclimatic and land use variables. Variables related to temperature and precipitation contributed to accurately predict the distribution of pure populations of each species, but the models did not perform well in areas where genetically admixed populations occur. A sliding window approach proved useful to identify different sets of variables that are important in different sections of this long and heterogeneous hybrid zone, and offers good potential to predict the fate of moving contact zones in global change scenarios. [ABSTRACT FROM AUTHOR]

Although the search for the drivers of amphibian declines continues, there is a need to implement conservation actions. Conservation science usually does not deliver clear answers about which conservation actions are most effective and which ones should be implemented. Furthermore, results often cannot be used directly by conservationists. Given that resources are limited, there is a need to know which conservation actions and management interventions are most likely to succeed. The goal of evidence-based conservation is to assess the effectiveness of conservation actions qualitatively and quantitatively, and comparative effectiveness studies are a powerful tool to evaluate different conservation actions. We use a case study on toad tunnels to discuss the benefits and limitations of comparative effectiveness studies. Although we show that wider tunnels are used by a higher proportion of individuals, the strength of evidence for effects of other characteristics of amphibian tunnels on tunnel use was weak. Despite some equivocal results, our case study illustrates that the approach can readily be used to study the effectiveness of conservation actions and to derive recommendations for conservationists and managers that can be used directly to improve future conservation interventions. [ABSTRACT FROM AUTHOR]

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

Road-based citizen science surveys are increasingly used for long-term monitoring of wildlife, including amphibians, over large spatial scales. However, how representative such data are when compared to the actual species distribution remains unclear. Spatial biases in site selection or road network coverage by volunteers could skew results towards more urbanised areas and consequently produce incorrect or partial trend estimations at regional or national scales. Our objective was to compare and verify potential spatial biases of road-based data using distribution datasets of different origins. We used as a case study the common toad (Bufo bufo), a fast-declining species and the main amphibian targeted by conservation action on roads in Europe. We used Maxent models to compare road survey data obtained from the 35 year-long "Toads on Roads" project in Great Britain with models using national-scale toad distribution records as well as with models using randomly generated points on roads. Distribution models that used data collected by volunteers on roads produced similar results to those obtained from overall species distribution, indicating the lack of selection bias and high spatial coverage of volunteer-collected data on roads. Toads were generally absent from mountainous areas and, despite the high availability of potential recorders, showed nearly complete absence of road-based records in large urban areas. This is probably the first study that comparatively evaluates species distribution models created using datasets of different origin in order to verify the influence of potential spatial bias of data collected by volunteers on roads. Large-scale declines of widespread amphibians have been demonstrated using data collected on roads and our results indicate that such data are representative and certainly comparable to other existing datasets. We show that for countries with high road network coverage, such as Great Britain, road-based data collected by volunteers represent a robust dataset and a critical citizen science contribution to conservation. [ABSTRACT FROM AUTHOR]

Genetic differentiation plays an integral role in species persistence. However, it remains challenging to quantify the ways in which the degree of isolation affects animal populations. The Common Toad (Bufo bufo) is a species of conservation concern, particularly in the UK, where populations have undergone large-scale declines. There are two types of isolation that are relevant to toad population persistence. These are: 1) isolation by distance (IBD), where populations that are further apart become more isolated with time; and 2) isolation by barrier (IBB), where the presence of barriers to movement can isolate populations. Both IBB and IBD are relevant to Common Toad survival, and thus conservation, in fragmented landscapes typical of farmlands. We collected Common Toad genetic material from eight different breeding ponds amongst the rural farmland landscapes of Oxfordshire, England to test the effect of IBD and IBB on toad genetic differentiation. We detected no significant effect of IBD (range 2–22 km between breeding ponds) on Common Toad genetic differentiation at this scale. We did, however, identify a significant and positive relationship between IBB and Common Toad genetic differentiation. Breeding populations were more genetically different with increasing barrier distance. The lack of a relationship between IBD and toad genetic differentiation could suggest that Common Toads are not as philopatric as previously thought, with reduced availability of suitable breeding ponds possibly driving more migrants to disperse greater distances and thereby possibly improving genetic mixing of the metapopulation. [ABSTRACT FROM AUTHOR]

We tracked yearly variation in breeding phenology in relation to weather parameters in a common European toad population from the southeastern part of its range. Phenological data were collected from 2001 to 2003 and from 2011 to 2017 and compared to open-access daily weather data from a nearby weather station. Data analysis revealed no significant effect of weather on initiation of breeding and a negative relationship between mean daily humidity and mean cloud cover on the duration of breeding season (P = 0.03). Further analysis showed a decreasing trend in breeding season humidity in the past 70 years. Our results predict a tendency toward longer toad breeding seasons in years with drier winter/spring. A projected decrease in humidity in this region could prolong toad breeding season, potentially exposing adults to higher predation. Therefore, the scenario of further decline in our study population should be considered and conservation measures planned accordingly. [ABSTRACT FROM AUTHOR]

Using micro-CT and 3D landmark-based geometric morphometrics, I investigated postmetamorphic shape variation in the skull of Bufo bufo and Bufo spinosus, two widespread European toad species with small phenotypic differences. Two ontogenetic series were compared, for a total of 58 individuals. They exhibited similar allometric growth patterns, characterised by cranial widening with relative shortening and dorsoventral compression. However, some interspecific shape divergence was observed, particularly among adults: a relatively shorter skull and a more dorsally extended snout distinguished B. spinosus from B. bufo. This disparity, which gives further support to species separation, can probably be ascribed to changes in the allometric trajectories, and seen in light of the evolutionary history of the two lineages. [ABSTRACT FROM AUTHOR]

Bufo bufo is a living example of evolutionary processes due to its numerous physiological and ecological adaptations. This is the first study to genetically characterize the TGF-β gene family in B. bufo at the genome-wide level, and a total of 28 TGF-β gene family homologs are identified. Physicochemical characteristics of TGF-β homologs exhibit a basic nature except for BMP1, BMP4, BMP10, BMP15, AMH, INHA, NODAL Modulator and TGFB1. Phylogenetic analysis divided the TGF-β gene family homologs into 2 major clades along with other vertebrate species. In domain and motif composition analysis, the gene structure for all TGF-β homologs exhibited homogeneity except BMP1. We have identified the TGF-β propeptide domain together with the TGF-β in all family homologs of TGF-β superfamily. Gene structure comparisons indicated that the TGF-β gene family have arisen by gene duplications. We also identified 10 duplicated gene pairs, all of which were detected to be segmental duplications. The Ka/Ks test ratio findings for every pair of genes revealed that none of the ratios surpassed 1 except for one gene pair (INHA/BMP1), indicating that these proteins are under positive selection. Circos analysis showed that TGF-β gene family homologs are arranged in 11 dispersed clusters and all were segmentally arrayed in the genome. This study provides a molecular basis for TGF-β ligand protein functional analysis and may serve as a reference for in-depth phylogenomics and may promote the development of novel strategies.Communicated by Ramaswamy H. Sarma.

This study explored the biosynthesis of bufadienolides(BDs) in Bufo bufo gargarizans to solve the dilemma of the decreasing resources of B. bufo gargarizans and provide a theoretical basis for the sustainable utilization of the resources. Ultra-high performance liquid chromatography-Orbitrap-mass spectrometry(UHPLC-Orbitrap-MS) was employed to detect the synthesis sites of BDs in B. bufo gargarizans, and the results were verified by desorption electrospray ionization-mass spectrometry imaging(DESI-MSI) and homogenate incubation experiments. BDs in B. bufo gargarizans had the highest content in the liver and the highest concentration in the gallbladder, in addition to the parotid gland and skin, which suggested that the liver could synthesize BDs. The results of DESI-MSI also showed that BDs were mainly enriched in the liver rather than the immature parotid gland. The incubation experiment of liver homogenates demonstrated the liver of B. bufo gargarizans had the ability to synthesize BDs. This study showed that the liver was a major organ for the synthesis of BDs in B. bufo gargarizans during metamorphosis, development, and growth, which provided strong theoretical support for the biosynthesis of BDs and the sustainable utilization of B. bufo gargarizans resources.