Popliteal sinus venipuncture in anurans.

OBJECTIVE: To demonstrate an underutilized, reliable, and technically simple anuran venipuncture technique by use of the popliteal sinus. ANIMALS: Medium to large (> 30 g) anuran species. METHODS: The animal is manually restrained using an encircling grip around the pelvic girdle. With the ventrum facing the phlebotomist, the phlebotomist grasps the distal hind limb and gently extends it. If necessary, the medial stifle can be rinsed with sterile water or saline to remove gross debris. Using a caudomedial approach, a 25- to 27-G, 0.5-inch (1.27-cm) needle and attached syringe is advanced toward the stifle at an approximately 45° angle while maintaining negative pressure. If lymphatic fluid or a visibly lymphatic fluid-contaminated blood sample is obtained, the sample is discarded and the attempt is repeated with a new needle and syringe. Collection volume should not exceed 1% of body weight in systemically healthy animals. In compromised animals, a reduction in collection volume should be considered (eg, ≤ 0.5% of body weight). While the phlebotomist may have an ergonomic preference, either hind limb can be used for blood collection. RESULTS: Popliteal sinus venipuncture in anurans can be safely and easily performed with only manual restraint. CLINICAL RELEVANCE: Commonly utilized anuran blood collection techniques include venipuncture of the ventral abdominal vein and lingual venous plexus and cardiocentesis. These techniques can be technically difficult and may pose risks to the patient, particularly if attempted without chemical restraint. Popliteal sinus venipuncture is a reliable, technically simple alternative blood collection technique that can be safely performed in anurans with only manual restraint.

Morphological and molecular data warrant the description of a new species of the genus Scutiger (Anura, Megophryidae) from the Central Himalaya.

Recent phylogenetic studies in Himalayan lazy toads of the genus Scutiger Theobald, 1868 revealed the presence of genetically deeply divergent lineages. The taxonomy of S.nepalensis sensu lato was re-assessed based on museum material considering molecular and morphological data. The results strongly support the recognition of a new species, S.kanjiroba sp. nov. distributed along the Nepalese Kanjiroba massif. It is further shown that S.sikimmensis has an apparently much more restricted distribution range than previously thought. The frequent misidentification of Scutiger across large areas of the Himalaya-Tibet area highlights the significance of careful taxonomic evaluation of collection material and the need for the direct morphological comparison of closely related species when describing new species.

Myiasis by the Toad Fly (Lucilia bufonivora; Calliphoidae) in Amphibians in Montana, USA.

Toad flies in the genus Lucilia (previously referred to as Bufolucilia spp.) parasitize and cause myiasis in several amphibian species in North America. From 2019 to 2022, we documented Lucilia bufonivora infections in post-metamorphic western toads (Anaxyrus boreas) during amphibian surveys in four wetlands in Glacier National Park, Montana, US. We found nine infected adult toads in 2019, seven infected adults in 2020, one infected juvenile in 2021, and five infected adults plus one infected juvenile in 2022. We also captured Columbia spotted frogs (Rana luteiventris) during these same surveys but detected no infections. Only one of the four wetlands had infected toads in 2019, despite their proximity and hydrologic connectivity, but two of these wetlands had infections in 2020, and a third had a single infection in 2021. The same three of four wetlands had infections in 2022. In 2008, a similar parasitic infection in one western toad had been noted at the same wetland as in 2019. That toad had been captured again two years later without signs of infection.

Combinational Antitumor Strategies Based on the Active Ingredients of Toad Skin and Toad Venom.

A multidrug combination strategy is an important mean to improve the treatment of cancer and is the mainstream scheme of clinical cancer treatment. The active ingredients of traditional Chinese medicine, represented by toad skin and toad venom, have the advantages of high efficiency, low toxicity, wide action and multiple targets and have become ideal targets in combined treatment strategies for tumors in recent years. Toad skin and toad venom are traditional Chinese animal medicines derived from Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider that have shown excellent therapeutic effects on the treatment of various cancers and cancer pain as adjuvant antitumor drugs in clinical practice. The involved mechanisms include inducing apoptosis, arresting the cell cycle, inhibiting cell proliferation, migration and invasion, inhibiting tumor angiogenesis, reversing the multidrug resistance of tumor cells, and regulating multiple signaling pathways and targets. Moreover, a multidrug combination strategy based on a nanodelivery system can realize the precise loading of the active ingredients of toad skin or toad venom and other antitumor drugs and carry drugs to overcome physiological and pathological barriers, complete efficient enrichment in tumor tissues, and achieve targeted delivery to tumor cells and the controlled release of drugs, thus enhancing antitumor efficacy and reducing toxicity and side effects. This article reviewed the clinical efficacy and safety of the combination of toad skin and toad venom with chemotherapeutic drugs, targeted drugs, analgesics and other drugs; evaluated the effects and mechanisms of the combination of toad skin and toad venom with chemotherapy, targeted therapy, radiotherapy or hyperthermia, traditional Chinese medicine, signaling pathway inhibitors and other therapies in cell and animal models; and summarized the codelivery strategies for the active ingredients of toad skin and toad venom with chemotherapeutic drugs, small-molecule targeted drugs, monoclonal antibodies, active ingredients of traditional Chinese medicine, and photodynamic and photothermal therapeutic drugs to provide a basis for the rational drug use of toad skin and toad venom in the clinic and the development of novel drug delivery systems.

Revisiting current distribution and future habitat suitability models for the endemic Malabar Tree Toad (Pedostibes tuberculosus) using citizen science data.

Climate change is one of the major drivers of biodiversity loss. Among vertebrates, amphibians are one of the more sensitive groups to climate change due to their unique ecology, habitat requirements, narrow thermal tolerance and relatively limited dispersal abilities. We projected the influence of climate change on an endemic toad, Malabar Tree Toad (Pedostibes tuberculosus; hereafter MTT) from the Western Ghats biodiversity hotspot, India, for two different shared socio-economic pathways (SSP) using multiple modeling approaches for current and future (2061-2080) scenarios. The data used predominantly comes from a citizen science program, 'Mapping Malabar Tree Toad' which is a part of the Frog Watch citizen science program, India Biodiversity Portal. We also evaluated the availability of suitable habitats for the MTT in Protected Areas (PAs) under the current and future scenarios. Our results show that annual precipitation was the most important bioclimatic variable influencing the distribution of MTT. We used MaxEnt (MEM) and Ensemble (ESM) modeling algorithms. The predicted distribution of MTT with selected environmental layers using MEM was 4556.95 km2 while using ESM was 18,563.76 km2. Overlaying PA boundaries on predicted distribution showed 37 PAs with 32.7% (1491.37 km2) and 44 PAs with 21.9% (4066.25 km2) coverage for MEM and ESM respectively. Among eight future climate scenarios, scenarios with high emissions showed a decreased distribution range from 33.5 to 68.7% of predicted distribution in PAs, while scenarios with low emissions showed an increased distribution range from 1.9 to 111.3% in PAs. PAs from the Central Western Ghats lose most suitable areas with a shift of suitable habitats towards the Southern Western Ghats. This suggests that MTT distribution may be restricted in the future and existing PAs may not be sufficient to conserve their habitats. Restricted and discontinuous distribution along with climate change can limit the dispersal and persistence of MTT populations, thus enhanced surveys of MTT habitats within and outside the PAs of the Western Ghats are an important step in safeguarding the persistence of MTT populations. Overall, our results demonstrate the use of citizen science data and its potential in modeling and understanding the geographic distribution and the calling phenology of an elusive, arboreal, and endemic amphibian species.

Morphology and vocalization comparison of the Houston Toad and the Dwarf American Toad: implications for their historic range.

Documenting changes in the distribution and abundance of a given taxon requires historical data. In the absence of long-term monitoring data collected throughout the range of a taxon, conservation biologists often rely on preserved museum specimens to determine the past or present, putative geographic distribution. Distributional data for the Houston Toad (Anaxyrus houstonensis) has consistently been confounded by similarities with a sympatric congener, the Dwarf American Toad (A. americanus charlesmithi), both in monitoring data derived from chorusing surveys, and in historical data via museum specimens. In this case, misidentification can have unintended impacts on conservation efforts, where the Houston Toad is federally endangered, and the Dwarf American Toad is of least concern. Previously published reports have compared these two taxon on the basis of their male advertisement call and morphological appearance, often with the goal of using these characters to substantiate their taxonomic status prior to the advent of DNA sequencing technology. However, numerous studies report findings that contradict one another, and no consensus on the true differences or similarities can be drawn. Here, we use contemporary recordings of wild populations of each taxon to test for quantifiable differences in male advertisement call. Additionally, we quantitatively examine a subset of vouchered museum specimens representing each taxon to test previously reported differentiating morphometric characters used to distinguish among other Bufonids of East-Central Texas, USA. Finally, we assemble and qualitatively evaluate a database of photographs representing catalogued museum vouchers for each taxon to determine if their previously documented historic ranges may be larger than are currently accepted. Our findings reveal quantifiable differences between two allopatric congeners with respect to their male advertisement call, whereas we found similarities among their detailed morphology. Additionally, we report on the existence of additional, historically overlooked, museum records for the Houston Toad in the context of its putative historic range, and discuss errors associated with the curation of these specimens whose identity and nomenclature have not been consistent through time. These results bookend decades of disagreement regarding the morphology, voice, and historic distribution of these taxa, and alert practitioners of conservation efforts for the Houston Toad to previously unreported locations of occurrence.

Insights into phylogenetic positions and distribution patterns: Complete mitogenomes of two sympatric Asian horned toads in Boulenophrys (Anura: Megophryidae).

Boulenophrys sangzhiensis and Boulenophrys tuberogranulata, two narrow-distributed toad species within the Megophryidae family in southern China, are experiencing population declines due to habitat loss and degradation. Despite their critical conservation status, the two species remain largely overlooked in public and scientific spheres. This study presented the first sequencing, assembly, and annotation of the complete mitogenomes of both species using next-generation sequencing. The mitogenome of B. sangzhiensis was 16,950 bp, while that of B. tuberogranulata was 16,841 bp, each comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a noncoding control region (D-loop). The gene content, nucleotide composition, and evolutionary rates of each mitogenome were analyzed. Both mitogenomes exhibited negative AT skew and GC skew with high A + T content. ATP8 exhibited the highest evolutionary rate, while COI had the lowest. A phylogenetic analysis based on 28 mitogenomes revealed two major clades of Megophryidae, supporting the classification of two subfamilies, Megophryinae and Leptobrachiinae. Within the subfamily Megophryinae, the genus Boulenophrys was divided into two species groups. Intriguingly, despite coexisting in Zhangjiajie City, B. sangzhiensis and B. tuberogranulata exhibited distinct origins from the two different species groups, underscoring the unique role of the coexisting area Zhangjiajie in driving their speciation and preserving their current populations. A parallel pattern was also identified in the Leptobrachiinae genus Leptobrachium within the same region. This study provided valuable data references and enhanced our understanding of the molecular characteristics of these threatened amphibian species.

Advances on Delivery System of Active Ingredients of Dried Toad Skin and Toad Venom.

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.

Spatially limited pathogen pollution in an invasive tick and host system.

Expansion of global commerce has facilitated pathogen pollution via the transportation and translocation of invasive species and their associated parasites and pathogens. In Florida, imported cane toads (Rhinella horribilis) were accidentally and intentionally released on multiple occasions. Early populations were found to be infested with the invasive tick, Amblyomma rotundatum, yet it is unknown if these ticks dispersed with their hosts as cane toads spread throughout much of the state. The objectives of our investigation were to (1) determine if there are fewer tick infestations on toads at the periphery than at the core of their distribution as predicted by founder effect events, and (2) identify if ticks were infected with exotic pathogens. We captured toads from 10 populations across Florida. We collected ticks, vent tissue, and tick attachment site tissue from each toad, then tested samples for bacteria in the genus, Rickettsia. We found that 3/10 populations had toads that were infested with A. rotundatum, and infested individuals were in the earliest introduced populations at the core of their distribution. Pathogen testing confirmed Rickettisa bellii in ticks, but not in toad tissues. Haplotype networks could not clearly distinguish if R. bellii in Florida was more closely related to North or South American strains, but host-tick associations suggest that the pathogen was exotic to Florida. Our investigation demonstrated that an invasive species facilitated the introduction of parasites and pathogens into Florida, yet the invasive tick species encountered limitations to dispersal on this host species. Supplementary Information: The online version contains supplementary material available at 10.1007/s10530-024-03291-9.

Bufadienolide-Fatty Acid Conjugates from the Fertilized Eggs of Toad Bufo gargarizans: Isolation, Characterization, Toxicity, and Antiproliferative Evaluation.

Bufadienolides (BDs) are a class of naturally occurring toxins present in amphibian toads. Serving as the chemical weapons, they exist not only in the adult toads but also in toad eggs. Guided by mass spectrometry (MS)-based component analysis and feature-based molecular networking (FBMN), 30 bufadienolide-fatty acid conjugates (BDFs) were isolated from the fertilized eggs of toad Bufo gargrizans, including 25 previously undescribed compounds (1-25). Their chemical structures were elucidated by extensive spectroscopic analysis, chemical methods, and GC-MS. The toxicities of all BDFs and their corresponding free BDs were assessed using the zebrafish model. The structure-toxicity relationship analysis showed that the modification of BDs by hydroxy fatty acids can cause a significant increase of the toxicity. Furthermore, all the isolated compounds were evaluated for their antiproliferative activities in pancreatic cancer cell lines ASPC-1 and PANC10.05. The structure-activity relationship (SAR) analysis revealed that BDFs with hellebrigenin as the bufogenin moiety (6 and 7) exhibited the most potent antiproliferative effect. Further investigation into their functional mechanism demonstrated that 6 and 7 induced apoptosis in pancreatic cancer cells PANC10.05 and significantly suppressed the expression of the apoptosis-related gene c-MYC. In addition, 6 and 7 effectively inhibited the expression of the PI3K/Akt/mTOR pathway in PANC10.05. Moreover, we assessed the efficacy of 6 and 7 on cancer cells from various tissues and observed their broad-spectrum antiproliferative activity.

The role of phenotypic plasticity and corticosterone in coping with pond drying conditions in yellow-bellied toad (Bombina variegata, Linnaeus 1758) tadpoles.

Amphibian larvae inhabiting temporary ponds often exhibit the capacity to accelerate development and undergo metamorphosis in challenging conditions like desiccation. However, not all species exhibit this ability, the yellow-bellied toad (Bombina variegata) is one such example. The underlying mechanisms behind the inability to accelerate development under desiccation remain largely unexplored. The hypothalamic-pituitary-interrenal (HPI) axis and corticosterone (CORT), which act synergistically with thyroid hormone, are thought to facilitate metamorphosis in response to desiccation stress. In this study, we aimed to investigate whether modification in the HPI axis, particularly CORT levels, contributes to the absence of adaptive plasticity in B. variegata under desiccation stress. The study design included four treatments: high water level, high water level with exogenous CORT, low water level, and low water level with metyrapone (a CORT synthesis inhibitor). The main objective was to evaluate the effects of these treatments on whole-body corticosterone levels, life history, morphological traits, and oxidative stress parameters during the prometamorphic and metamorphic climax developmental stages. While low water level had no effect on total corticosterone levels, larval period, body condition index, and metamorphic body shape, it negatively affected metamorph size, mass, and growth rate. Our findings suggest that constant exposure to desiccation stress over generations may have led to modifications in the HPI axis activity in B. variegata, resulting in adaptation to changes in water level, evident through the absence of stress response. Consequently, CORT may not be a relevant stress indicator in desiccation conditions for this species.

Isolation, Identification and Chemical Modification of Bufadienolides from Bufo melanostictus Schneider and Their Cytotoxic Activities against Prostate Cancer Cells.

The traditional Chinese medicine toad venom (Venenum bufonis) has been extensively used to treat various diseases, including cancers, in China and other Southeast Asian countries. The major constituents of toad venom, e.g., bufadienolides and alkaloids, exhibit broad-spectrum pharmacological effects in cancers. Herein, two new bufadienolides (1 and 2), along with eleven known compounds (3-13) were successfully isolated from Bufo melanostictus Schneider. Their structures were elucidated by extensive spectroscopic data and X-ray diffraction analysis. Furthermore, four lactam derivatives were synthesized through the transformation of bufadienolides lactones. The inhibitory effects of these compounds against human prostate cancer cell lines PC-3 and DU145 were evaluated. The outcomes indicated a notable trend, with a substantial subset displaying nanomolar range IC50 values against PC-3 and DU145 cells, underscoring their pronounced cytotoxicity. Moreover, a noteworthy distinction surfaces, wherein lactones consistently outperformed their lactam counterparts, further validating their heightened potency for the treatment of prostate cancer. This study contributes significant preclinical evidence substantiating the therapeutic viability of bufadienolides and toad venom as intervention strategies for prostate cancer.

Evolution of air-borne vocalization: Insights from neural studies in the archeobatrachian species Bombina orientalis.

Vocalization of tetrapods evolved as an air-driven mechanism. Thus, it is conceivable that the underlaying neural network might have evolved from more ancient respiratory circuits and be made up of homologous components that generate breathing rhythms across vertebrates. In this context, the extant species of stem anurans provide an opportunity to analyze the connection of the neural circuits of lung ventilation and vocalization. Here, we analyzed the fictive lung ventilation and vocalization behavior of isolated brains of the Chinese fire-bellied toad Bombina orientalis during their mating season by nerve root recordings. We discovered significant differences in durations of activation of male brains after stimulation of the statoacoustic nerve or vocalization-relevant forebrain structures in comparison to female brains. The increased durations of motor nerve activities in male brains can be interpreted as fictive calling, as male's advertisement calls in vivo had the same general pattern compared to lung ventilation, but longer duration periods. Female brains react to the corresponding stimulations with the same shorter activity pattern that occurred spontaneously in both female and male brains and thus can be interpreted as fictive lung ventilations. These results support the hypothesis that vocal circuits evolved from ancient respiration networks in the anuran caudal hindbrain. Moreover, we could show that the terrestrial stem archeobatrachian Bombina spec. is an appropriate model to study the function and evolution of the shared network of lung ventilation and vocal generation.

Is developmental plasticity triggered by DNA methylation changes in the invasive cane toad (Rhinella marina)?

Many organisms can adjust their development according to environmental conditions, including the presence of conspecifics. Although this developmental plasticity is common in amphibians, its underlying molecular mechanisms remain largely unknown. Exposure during development to either 'cannibal cues' from older conspecifics, or 'alarm cues' from injured conspecifics, causes reduced growth and survival in cane toad (Rhinella marina) tadpoles. Epigenetic modifications, such as changes in DNA methylation patterns, are a plausible mechanism underlying these developmental plastic responses. Here we tested this hypothesis, and asked whether cannibal cues and alarm cues trigger the same DNA methylation changes in developing cane toads. We found that exposure to both cannibal cues and alarm cues was associated with local changes in DNA methylation patterns. These DNA methylation changes affected genes putatively involved in developmental processes, but in different genomic regions for different conspecific-derived cues. Genetic background explains most of the epigenetic variation among individuals. Overall, the molecular mechanisms triggered by exposure to cannibal cues seem to differ from those triggered by alarm cues. Studies linking epigenetic modifications to transcriptional activity are needed to clarify the proximate mechanisms that regulate developmental plasticity in cane toads.

3α-Hydroxybufadienolides in Bufo gallbladders: structural insights and biotransformation.

Bufadienolides, naturally occurring steroids primarily found in toads, have garnered attention for their pharmacological properties and ecological significance. In this study, we isolated and identified 21 bufadienolides from the gallbladders of Bufo gargarizans, comprising four new compounds and 17 known ones. Notably, the predominance of 15 bufadienolides with a 3α-OH configuration in toad bile differs significantly from the 3ß-OH bufadienolides found in venom secreted by toad glands. Moreover, our investigation into the biotransformation of 3ß-OH and 3α-OH bufadienolides in the liver and kidney tissues of toads revealed an irreversible conversion from 3ß-OH to 3α-OH bufadienolides, suggesting a crucial role in toad self-detoxification. These findings provide valuable insights into the structural diversity of bufadienolides and advance our understanding of their medical and ecological significance.

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.

Integrated post-acquisition data processing strategy for rapid steroid sulfate characterization in Toad gall-bladder.

LC-MS serves as a workhorse for chemical profile characterization of Chinese medicinal materials (CMMs) attributing to the ability of measuring fruitful MS/MS spectral information. However, it is laborious to extract the information belonging to the compounds-of-interest from the massive data matrixes even employing those well-defined post-acquisition data processing strategies. Here, efforts were devoted to propose an integrated strategy allowing rapid chemical homologs-focused data filtering through integrating the fit-for-purpose existing strategies, such as molecular weight imprinting (MWI), diagnostic fragment ion filtering (DFIF), neutral loss filtering (NLF), and isotope pattern filtering (IPF). Homologs-focused chemical characterization of a precious CMM namely Toad gall-bladder (Chinese name: Chandan) that is rich of diverse effective steroid sulfates, particularly bufogenin sulfates, bile acid sulfates and bilichol sulfates, was employed as a proof-of-concept. Recombinant human SULT2A1-catalyzed in vitro metabolism was undertaken to generate eight bufogenin sulfates to facilitate summarizing MS/MS spectral behaviors. After in-house data library construction and MS1 and MS2 spectral acquisition, data filtering was conducted as follows: 1) MWI and IPF was utilized in combination to capture deprotonated molecular ions and the 34S isotopic ions for the sulfates of those reported steroids; 2) m/z 79.9568 (SO3-·) and 96.9596 (HSO4-) were applied to DFIF; and 3) SO3 (79.9568 Da) served as the feature to achieve NLF. Those captured MS/MS information subsequently participated in tentatively structural annotation through applying those empirical mass fragmentation rules. As a result, 71 compounds including 7 bufogenin sulfates, 17 bile acid sulfates, 13 bilichol sulfates and a C-23 steroid sulfate were detected from Toad gall-bladder and thereof, 39 ones received plausible identities assignment. Above all, the steroid sulfates in Toad gall-bladder were profiled in depth, and more importantly, the proposed strategy should be a meaningful option for, but not limited to, submetabolome characterization in CMMs.

Exploring peptides from toad venom for source identification by LC-MS/MS using MRM method.

Toad venom is a traditional Chinese medicine (TCM) with various sources and wide-ranging preparations. Previous quality assessment studies primarily concentrated on small molecular compounds like toad dienolactones and indole alkaloids, studies on macromolecular peptides and proteins as quality assessment standards remained at the qualitative stage, lacking the development of practical and convenient quantitative methods. In this study, to explore the peptides from toad venom as a new method for identifying and evaluating its source, a complete scan of the water extract of peptides from toad venom was conducted using HPLC-Quadrupole Time-of-Flight Mass Spectrometer (Q-TOF) 5600, leading to the identification of peptides based on mass spectrometry data. Subsequently, HPLC- Quadrupole-Linear Ion Trap Mass Spectrometer (Q-Trap) 5500 employing Multiple Reaction Monitoring (MRM) mode was utilized to quantitatively analyze peptides in various sources of toad venom, followed by Partial Least Squares Discriminant Analysis (PLS-DA) to further analyze the data and evaluate the effectiveness. This study highlights the importance of exploring macromolecular substance in natural products research and provides a foundation for further studies on toad venom.

Investigation of crude methanolic extract from poison secreted by the Rhaebo gutattus on status redox antioxidant in mice

The study evaluated the antioxidant properties of a crude methanolic extract (CME) from Rhaebo guttatus poison in mice over a period of 7 and 30 days. The mice were divided into groups and treated with different concentrations of the extract (0; 8 µg mL-1; 16 µg mL-1 and 32 µg mL-1 or vehicle; 100 µL/animal/day; via gavage). The liver samples were analyzed for status redox parameters as catalase (CAT), glutathione-S-transferase (GST), reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS). The results showed that the CME caused changes in the levels of various antioxidants and oxidative stress markers. At 7 days, there was an increase in TBARS levels (8 µg mL-1 dose) and GST activity (16 µg mL-1 dose), and a reduction in GSH levels (32 µg mL-1 dose) compared to the control group. At 30 days, TBARS and GSH levels returned to control values in the same period, but GSH increased (32 µg mL-1 dose) compared at 7 days; GST activity remained high after 30 days for 32 µg mL-1 dose compared other groups and time of treatment (7 days). Overall, the study suggests that the extract modulates antioxidant properties per se that can affect various markers of status redox in the liver of mice, mainly 16 µg mL-1 dose demonstrated to act under antioxidant enzymes in different times (7 or 30 days).

High frequency of larval abnormalities of the toad Rhinella arenarum (Anura: Bufonidae) in an Argentinian agroecosystem

Introduction: The frequent use of pesticides is currently considered a cause of environmental pollution due to the high rate of entry of these substances into agroecosystems. This constitutes a risk for the species that inhabit these ecosystems, in particular anurans whose characteristics make them prone to exposure to and interaction with environmental pollutants. Objective: To report the occurrence of abnormalities in larvae of the common toad Rhinella arenarum inhabiting ponds surrounded by agroecosystems. Methods: In two consecutive springs (2015 and 2016), reproductive events of common toads were monitored in temporary pond systems in agricultural and non-agricultural areas, located near the city of La Plata (Buenos Aires, Argentina). The physicochemical parameters of the ponds were measured, and the stage of each reproductive event was recorded, such as the numbers of adult toads, amplexus and clutches. In the laboratory, the larvae were measured and photographed, their stage of development was recorded, and their morphology was examined under a stereomicroscope. Representative samples (normal and abnormal) from each pond studied were processed for histopathological analysis. Results: In the field studies carried out on a population of R. arenarum collected in an agroecosystem, a lower number of reproductive adults and clutches were observed in relation to the population of a non-agricultural pond. A total of 1 910 larvae were collected: 529 and 1 381 larvae from ponds located in non-agricultural and agricultural areas, respectively. Larvae from the agroecosystem showed two types of abnormalities: severe tail flexure and abdominal bloating. In addition, five degrees of severity could be determined in relation to abdominal bloating. Conclusions: This work reports the high frequency and severity of abnormalities observed in the early stages of R. arenarum larvae living within an agroecosystem, providing evidence of the negative impact that agricultural activities cause on aquatic ecosystems surrounded by farming areas.

Introducción: El uso frecuente de plaguicidas es considerado actualmente una causa de contaminación ambiental debido a las altas tasas de ingreso de estas sustancias a los agroecosistemas. Esta situación es un riesgo para las especies que habitan en estos ecosistemas, en particular los anuros cuyas características los hacen propensos a la exposición e interacción con contaminantes ambientales. Objetivo: Informar la presencia de anormalidades en larvas del sapo común Rhinella arenarum que habitan en estanques rodeados por un agroecosistema. Métodos: En dos primaveras consecutivas (2015 y 2016), se monitorearon los eventos reproductivos del sapo común proveniente de sistemas de estanques temporales ubicados en zonas agrícolas y no agrícolas, cerca de la ciudad de La Plata (Buenos Aires, Argentina). Se midieron los parámetros fisicoquímicos de los estanques y se registraron las etapas de cada evento reproductivo como el número de sapos adultos, amplexos y nidadas. En el laboratorio, las larvas fueron medidas y fotografiadas, se registró su estado de desarrollo y se examinó la morfología de cada una bajo microscopio estereoscópico. Se procesaron muestras representativas (normales y anormales) de cada estanque estudiado para análisis histopatológico. Resultados: En la población de R. arenarum que vive dentro de un agroecosistema, se observó un menor número de adultos reproductores y puestas en relación con la del estanque en la zona no agrícola. Se recolectaron un total de 1 910 larvas: 529 y 1 381 larvas de estanques ubicados en zonas no agrícolas y agrícolas, respectivamente. Las larvas del agroecosistema mostraron dos tipos de anormalidades: severa flexión de la cola y distensión abdominal. Además, se pudo determinar cinco grados de gravedad en relación con la distensión abdominal. Conclusiones: Una alta frecuencia y severidad de anormalidades en los estadios tempranos de larvas de R. arenarum que viven dentro de un agroecosistema proporciona evidencia del impacto negativo que las actividades agrícolas causan en los ecosistemas acuáticos rodeados por áreas de cultivo.