Species Of Bufo Research Articles

Comprehensive Analysis of Bufadienolide and Protein Profiles of Gland Secretions from Medicinal Bufo Species.

Toad Venom (TV) is the dried product of toxic secretions from Bufo bufo gargarizans Cantor (BgC) or B. melanostictus Schneider (BmS). Given the increasing medical demand and the severe depletion of wild toads, a number of counterfeit TVs appeared on the market, posing challenges to its quality control. In order to develop an efficient, feasible, and comprehensive approach to evaluate TV quality, a thorough analysis and comparison of chemical compounds among legal species BgC and BmS, as well as the main confusion species B. andrewsi Schmidt (BaS) and B. raddei Strauch (BrS), were conducted by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), high performance liquid chromatography (HPLC), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Nano LC-MS/MS analyses. We identified 126 compounds, including free or conjugated bufadienolides, indole alkaloids and amino acids, among the four Bufo species. The content of main bufadienolides, such as gamabufotalin, bufotalin, bufalin, cinobufagin, and resibufogenin, and the total protein contents varied widely among 28 batches of TV due to their origin species. The sum of the five bufadienolides within the BgC, BmS, BaS, and BrS samples were 8.15-15.93%, 2.45-4.14%, 11.15-13.50%, and 13.21-14.68%, respectively. The total protein content of BgC (6.9-24.4%) and BaS (19.1-20.6%) samples were higher than that of BmS (4.8-20.4%) and BrS (10.1-13.7%) samples. Additionally, a total of 1357 proteins were identified. There were differences between the protein compositions among the samples of the four Bufo species. The results indicated that BgC TV is of the highest quality; BaS and BrS TV could serve as alternative resources, whereas BmS TV performed poorly overall. This research provides evidence for developing approaches to evaluate TV quality and selecting the proper Bufo species as the origin source of TV listed in the Chinese pharmacopoeia.

Аналіз спектру живлення жаби трав’яної (Rana temporaria) та ропухи сірої (Bufo bufo) в антропогенно-трансформованих оселищах Українського Розточчя

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.

Genetic data to describe the hybrid zone between Bufo bufo (Linnaeus, 1758) and Bufo verrucosissimus (Pallas, 1814) in northeastern Türkiye

Abstract Hybrid zones are geographic areas where individuals from distinct taxa meet, mate, and hybridize. These zones may have complex histories, but many of them originated relatively recently, during climatic oscillations in the Quaternary, following range shifts of formerly isolated, well-differentiated lineages. The Bufo bufo species group comprises four species distributed over the Western Palearctic. Whereas the contact zone between Bufo bufo and B. spinosus in western Europe has been well characterized, little is known about other species contacts. Here we focused on the contact between B. verrucosissimus and B. bufo in northeastern Türkiye, using mtDNA and microsatellite markers to describe genetic structure and patterns of admixture in the hybrid zone based on Bayesian clustering and cline analyses. Both species meet in a narrow contact zone at Rize province, with restricted introgression suggesting barriers to hybridization consistent with species status. Spatial population genetic analyses of microsatellite data pinpoint a possible enclave population of B. bufo at the Borçka district in Artvin province, isolated within the B. verrucosissimus range. The centers of the microsatellite and mtDNA-based clines are slightly displaced, with B. verrucosissimus mtDNA introgressing about 33 km W of the nuclear contact. Hybrid zone dynamics seem to be associated with range shifts mediated by Pleistocene glacial cycles and/or sex-biased dispersal.

Antimicrobial Compounds from Skin Secretions of Species That Belong to the Bufonidae Family.

Skin secretions of toads are a complex mixture of molecules. The substances secreted comprise more than 80 different compounds that show diverse pharmacological activities. The compounds secreted through skin pores and parotid glands are of particular interest because they help toads to endure in habitats full of pathogenic microbes, i.e., bacteria, fungi, viruses, and protozoa, due to their content of components such as bufadienolides, alkaloids, and antimicrobial peptides. We carried out an extensive literature review of relevant articles published until November 2022 in ACS Publications, Google Scholar, PubMed, and ScienceDirect. It was centered on research addressing the biological characterization of the compounds identified in the species of genera Atelopus, Bufo, Duttaphrynus, Melanophryniscus, Peltopryne, Phrynoidis, Rhaebo, and Rhinella, with antibacterial, antifungal, antiviral, and antiparasitic activities; as well as studies performed with analogous compounds and skin secretions of toads that also showed these activities. This review shows that the compounds in the secretions of toads could be candidates for new drugs to treat infectious diseases or be used to develop new molecules with better properties from existing ones. Some compounds in this review showed activity against microorganisms of medical interest such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Coronavirus varieties, HIV, Trypanosoma cruzi, Leishmania chagasi, Plasmodium falciparum, and against different kinds of fungi that affect plants of economic interest.

Ladies in armor: A micro-computed tomographic study of skin calcification in European toads (genus Bufo).

Micro-computed tomography is a powerful tool toward the detailed reconstruction of internal and external morphology, in particular for ossified and other dense tissues. Here, we document and compare the level of calcification in the skin of the head and the parotoids (the external skin glands) in males and females of common and spined toads, Bufo bufo and B. spinosus. In some anurans, including Bufo species, a specific acellular calcified tissue layer within the dermis has been documented (the Eberth-Katschenko, or EK-layer). By a combination of micro-computed tomography and classical histology, we detected additional calcium deposits located in the dermal layer stratum spongiosum, positioned above the EK-layer. We showed that the level of calcification and the presence of additional calcium deposits are size and sex related, increasing in the order B. bufo males, B. spinosus males, B. bufo females to B. spinosus females. The last of these groups is the least variable. Bufo spinosus females have dense calcium deposits in the parotoids and the dorsal and ventral skin. Three-dimensional volume renderings and cross-sectional slices obtained by micro-CT scanning indicate that this approach is a promising technique for further studies on bufonid skin anatomy and geographic variation in skin calcification.

Bufo Rana in Homeopathy: Potentization, Toxicology, Clinical Consequences, and Therapeutic Use

Abstract Homeopathy, an alternative treatment method based on the principle of “like cures like,” has gained attention in recent years. This paper focuses on Bufo Rana, a homeopathic remedy prepared from the poison of the Bufo toad. We explore the process of potentization and dosage in homeopathy, discussing the various potencies and the influence of Avogadro’s number. Furthermore, we provide an overview of Bufo species, their historical depictions in art, and the toxicological properties of bufotenine, tryptamine, and bufotoxin found in Bufo toads. The clinical consequences of the Bufo species’ venom, including neurological manifestations and cardiac arrhythmias, and the therapeutic use of Bufo Rana in mental and neurological conditions are discussed. We summarize Bufo Rana’s symptoms and therapeutic relationships based on William Boericke’s materia medica. Further research is warranted to investigate the efficacy and safety of Bufo Rana and its role in treating specific conditions.

Intra- and Interspecific and Elevational Variation in Keratinized Spines on the Head in Two Closely Related Bufo Species

Intra- and Interspecific and Elevational Variation in Keratinized Spines on the Head in Two Closely Related Bufo Species

Phylogeography of Rhabdias spp. (Nematoda: Rhabdiasidae) collected from Bufo species in Honshu, Shikoku, and Kyushu, Japan including possible cryptic species.

The genus Rhabdias Stiles & Hassall, 1905 comprises lung parasites of amphibians and reptiles worldwide. In Japan, 9 species have been recorded, including Rhabdias incerta Wilkie, 1930 which has been reported only in Bufo species. In this study, to assess the diversity of R. incerta, we performed molecular analyses of Rhabdias species sampled from three species/subspecies of Japanese toads namely Bufo japonicus, B. japonicus formosus, and B. torrenticola, collected in various regions of Honshu, Shikoku, and Kyushu, Japan. DNA sequence divergence was compared using mtDNA (COI) and nuclear DNA (28S) to identify possible cryptic species. Morphological analysis was performed through light microscopy and scanning electron microscopy (SEM). The results revealed that Bufo spp. serve as hosts for at least three Rhabdias species. Morphologically, most samples were identified as R. incerta but with a longer body and larger buccal cavity than originally described. Rhabdias incerta appears to be specific to the genus Bufo and is further subdivided into two or three phylogroups based on subspecies divisions and biogeography of their host. Some Rhabdias specimens collected in this study resemble R. tokyoensis Wilkie, 1930, parasitic in hosts from the order Caudata, which suggests host switching. Both molecular and morphological analyses suggested the presence of undescribed and cryptic Rhabdias species within toads collected in Japan. This study was the first to molecularly characterize Rhabdias species in Japan, including novel sequences of R. incerta and two undescribed species.

Incorporation of latitude-adjusted bioclimatic variables increases accuracy in species distribution models

Latitudinal variations in species niche due to factors such as phenotypic plasticity and local adaptation pose fundamental problems for ecological niche modelling at large geographic scales. Species adaptation to varying climates across latitudes thus necessitates the latitudinal adjustment of climatic variables for incorporation into more accurate models of habitat suitability and species distribution. Previous studies have identified changes in species’ elevational distributions across latitudes, however few studies incorporate latitude-adjusted variables into suitability or distribution models. In this study, we utilized generalized additive models (GAMs) to adjust bioclimatic variables by latitude in order to remove latitudinal variation in ecological niche models. We incorporated these latitude-adjusted variables into maximum entropy (MaxEnt) models of two species of toad, Bufo sachalinensis (previously Bufo gargarizans) and Bufo stejnegeri, in northeast Asia. We additionally identified thresholds in bioclimatic and terrain variables that delineate boundaries between the two Bufo species where their ranges overlap. We found that GAM adjustment of bioclimatic variables effectively removed latitudinal variation in said variables. MaxEnt models incorporating these latitude-adjusted variables had significantly improved model fit (AUC and TSS) for both Bufo species. We identified a potential sympatric area shared by the two species and delineated a geographic boundary thresholding the probable presence of one species over the other. This study shows a clear improvement for distribution models with the inclusion of latitude-adjusted bioclimatic variables for both narrow and wide-ranging species. This has implications for future distribution modelling practices, especially for geographically widespread species, and has potential for modelling species distributions in climate change scenarios. Further, the methodology for delimiting species thresholds has future use in modelling contact zones of related species.

A simple PCR-RFLP method for identification and detection of medicinal toad and its crude drug even with serious degradation of DNA

The secretions of the venom glands, dry skin and whole body of several Bufo species have been used as traditional medicines in East Asia to treat heart failure and cancer. Due to the highly similar morphological features of Bufo species and their derived commercial crude drugs, along with the high similarity of chemical composition of the secretions of venom glands, it is very challenging to identify the medicinal toads and the related crude drugs. The cyt-b sequences provide useful information to authenticate medicinal Bufo species. Based on the cyt-b sequences, a simple PCR-RFLP method was established for the identification of the medicinally used Bufo species as well as their derived crude drugs. The 23 specimens from three medicinally used Bufo species, B. bufo gargarizans (Bbg), B. melanostictus (Bm) and B. raddei (Br), were clearly divided into 3 groups according to the sequences of amplified cyt-b regions, which could be digested by specific restriction enzymes NcoI, EcoRV and BstXI, respectively. Then the specific PCR-RFLP method was further used to identify 9 samples of commercial crude drugs even with serious degradation of DNA, and all nine samples were identified as B. bufo gargarizans. The method is suitable for identification of medicinally used Bufo species and the related crude drugs.

Antimicrobial Secretions of Toads (Anura, Bufonidae): Bioactive Extracts and Isolated Compounds against Human Pathogens.

Species of the family Bufonidae, better known as true toads, are widespread and produce bioactive substances in the secretions obtained from specialized skin macroglands. Some true toads have been employed as a folk remedy to treat infectious diseases caused by microbial pathogens. Recent publications based on in silico analysis highlighted the Bufonidae as promising sources of antimicrobial peptides. A review of the literature reveals that Bufonidae skin secretion extracts show inhibitory activity in vitro against clinical isolates of bacteria, resistant and standard strains of bacterial, and fungal and parasitic human pathogens. Secondary metabolites belonging to the classes of alkaloids, bufadienolides, and peptides with antimicrobial activity have been isolated from species of the genera Bufo, Bufotes, Duttaphrynus, and Rhinella. Additionally, some antimicrobial extracts and purified compounds display low cytotoxicity against mammal cells.

Taxonomic assessment and distribution of common toads (Bufo bufo and B. verrucosissimus) in Turkey based on morphological and molecular data

Abstract The Bufo bufo species group includes four species distributed in the western Palearctic: B. bufo, B. eichwaldii, B. spinosus and B. verrucosissimus. Both B. bufo and B. verrucosissimus are known to occur in Turkey, but their range boundaries and the taxonomic status of B. verrucosissimus are still uncertain. In this study, we analyzed the variation in a set of morphological characters and in two mitochondrial and two nuclear DNA markers to address these questions. Phylogenetic analyses of sequence data support two main clades of common toads in Turkey, corresponding to B. bufo and B. verrucosissimus. The latter is subdivided into two allopatric subclades including populations along the Mediterranean and Black Sea coast, respectively. Discriminant analysis of morphological data showed separation among groups as defined by molecular analyses. We discuss these results and their implications for the evolutionary history of common toads in Turkey.

Two New Cryptic Endemic Toads of Bufo Discovered in Central Nevada, Western United States (Amphibia: Bufonidae: Bufo [Anaxyrus

We describe two new cryptic species of Bufo within the subgenus Anaxyrus discovered in Central Nevada of the western United States. Our analyses revealed that these two localized endemic toads are genetically divergent and morphologically distinct, yet were concealed under the range of the broadly distributed western toad (Bufo boreas), which occurs throughout Nevada. The newly discovered species are close in geographic proximity to each other (albeit, in different hydrological basins) but have evolved unique morphological characters that are distinct from each other and distinctive from all allied taxa within the B. boreas species complex. The delimiting of these two rare toads emphasizes the link between taxonomic crypsis and inadequate conservation as these newly described species are vulnerable to extinction due to severely restricted geographic ranges, unknown population sizes, and dependency on rare, fragile wetland habitat, which is a limited resource within Nevada, the primary state that makes up the arid Great Basin. These two endemics join the Great Basin B. boreas species complex as imperiled new members, and our study demonstrates that our knowledge of anuran diversity is incomplete and that new discoveries can still be made, even in unlikely settings.

Phylogenetic Systematics of the Water Toad (Bufo stejnegeri) Elucidates the Evolution of Semi-aquatic Toad Ecology and Pleistocene Glacial Refugia

The water toad (Bufo stejnegeri) is endemic to Northeastern Asia (South Korea, North Korea, and China) and has unique ecology for a toad by being semi-aquatic and breeding in lotic environments. We use a suite of phylogenetic analyses to understand the evolution of B. stejnegeri’s distinctive ecology and the impact of Pleistocene glacial cycles on the biodiversity of Northeast Asia. For the evolution of a semi-aquatic lifestyle, although B. stejnegeri is closely related to two other semi-aquatic Bufo species (B. torrenticola and B. andrewsi), ancestral state reconstruction analysis infers an independent evolution in all three species. Upon closer inspection, B. stejnegeri exhibits major differences in amplexus and egg-laying behaviour compared to the other two species, supporting independent evolution. Divergence dating analyses infer B. stejnegeri to have originated during the Late Miocene-Early Pliocene (4.3 Ma, 2.7–6.2 Ma). This species does not exhibit population differentiation with respect to mountain range, but shows a genetic pattern of southern richness and northern purity supporting a single refugium in Korea during Pleistocene glacial cycles. The Bayesian skyline plot supports this inference, inferring a population decline followed by expansion during the Pleistocene. Although not as species rich as the tropics, we hope this study helps spark interest in Northeast Asian biodiversity.

Karyotypic Analysis of the Spined Toad, <i>Bufo spinosus</i> Daudin, 1803 (Amphibia: Bufonidae)

For the first time, somatic chromosomes of Bufo spinosus were described (2 n = 22). It was close to the karyotypes of other West-Palearctic species of the genus Bufo and consisted of 10 large metacentric ( V ), 2 large submetacentric chromosomes ( SV ), 8 small metacentric ( v ), and 2 small submetacentric chromosomes ( sv ): 10 V + 2 SV + 8 v + 2 sv , NF = 44. All chromosomes of B. spinosus were characterized by presence of heterochromatin blocks in centromeric regions. No Q-stained bright bands were found. Ag-staining detected a nucleolar organizer region in the end of the long arm of the sixth pair of chromosomes. In all males studied, a dimorphism was found in the sixth pair of chromosomes. One chromosome in this pair was smaller than the second one. Differences in position of nucleolar organizer region or centromeric index between these chromosomes were not revealed.

Cloning, prokaryotic expression and function of the Bufo bufo gargarizans 3β-hydroxysteroid dehydrogenase (3βHSD) gene

Bufadienolides, one kind of steroids, are the major active component secreted by ear-side gland of Bufo species. Preliminary studies on high-throughput transcriptome sequencing about B. bufo gargarizans showed that the expression of 3β-Hydroxysteroid dehydrogenase (3βHSD) in ear-side gland was nearly 20 times higher than that in liver. The enzyme 3βHSD is an essential step in the biosynthesis of steroid such as progesterone, estrogens and androgens in steroidogenic tissues. Accordingly, 3βHSD is probably an important enzyme involved in the biosynthesis of bufadienolides. In this study, Bbg-3βHSD cDNA was cloned from the ear-side gland of B. bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pCOLD-Bbg3βHSD which was introduced into E. coli BL21 for prokaryotic expression. Bbg-3βHSD has an open reading frame (ORF) of 1134 bp and encodes 377 amino acid residues. The speculated protein molecular weight is 42.8 kDa and its theoretical isoelectric point is 8.68. Amino acid sequence homologous analysis showed that Bbg-3βHSD was highly homologous to the 3βHSD protein of other species. Phylogenetic tree showed the highest similarity between Bbg-3βHSD and 3βHSD from Rana rugosa. The optimized expression of recombinant Bbg-3βHSD were achieved by inducing with 0.1 mmol L−1 IPTG at 15 °C for 20 h. Enzymatic activity in vitro shows that pregnenolone and dehydroepiandroesterone could be 3β-oxidized by Bbg-3βHSD when NAD+ was used as the coenzyme. Enzymatic properties showed that the optimum reaction temperature of recombinant Bbg-3βHSD was 40 °C, the optimum pH was 8.5, and the optimum coenzyme concentration was 1.5 mmol L−1.

Characterization of the Adaptive Morphology of Japanese Stream Toad (Bufo torrenticola) Using Geometric Morphometrics.

The order Anura (frogs and toads) is a group of amphibians and contains over 6500 extant species living in a variety of environments. Each frog species evolved body form adaptive for living and breeding in their own habitats. In Japan, four taxa of Bufo are living: Western-Japanese common toad (Bufo japonicus japonicus), Eastern-Japanese common toad (B. japonicus formosus), Miyako toad (B. gargarizans miyakonis) and Japanese stream toad (B. torrenticola). The former three taxa breed in still water as many other species of Bufo do. In contrast, B. torrenticola breeds in running water such as streams in mountainous area. Corresponding to their breeding in a stream environment, both adult and larva of B. torrenticola acquired unique morphological characters. However, few study have explored differences in the body form between B. torrenticola and closely-related Bufo species quantitatively, remaining the details about the morphological adaptation to a stream environment in this toad species poorly understood. In this study, we quantitatively compared the shape of the foot and skull between the adult male of B. torrenticola and its close relative B. j. formosus using landmark-based geometric morphometrics. Our analyses revealed that B. torrenticola has relatively longer toe phalanges with relatively larger foot webs and relatively shorter metatarsals and a narrower and more streamlined skull, compared to closely-related B. j. formosus. These morphological characteristics are considered adaptive for their breeding in mountain torrents.

PPDPF cDNA Cloning from Two Bufo Species and Tissue Expression in B. gargarizans

PPDPF cDNA Cloning from Two Bufo Species and Tissue Expression in B. gargarizans

A New Species of Bufo (Amphibia, Anura) from the Miocene of Russia

A new species of Bufo is described from the Tagay locality (Irkutsk oblast’, Russia; late Early – early Middle Miocene). The material comprises disarticulated skull and postcranial bones. The described species can be differentiated from the other species of the genus primarily by the lateral depression of prootic and anterior position of the postorbital crest. The new taxon is similar to B. raddei in the ilium morphology and possibly belongs to the same radiation. The described species is the first fossil Bufo from the Early-Middle Miocene of Russia, and one of the oldest in Asia.

The complete mitochondrial genome of the Bufo stejnegeri (Anura: Bufonidae)

The complete mitochondrial genome of Bufo stejnegeri was determined, which was 17 939 bp in length. It consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one displacement loop (D-loop). The total length of D-loop region is 2533 bp, some tandem repeat units were found in this region. The phylogenetic trees of the 20 species from anura were reconstructed based on complete mtDNA sequences by Bayesian inference and maximum likelihood analyses. The result demonstrated that B. stejnegeri is the most closely related species with other Bufo species.