Bufo Gargarizans Research Articles

Light/dark phase influences intra-individual plasticity in maintenance metabolic rate and exploratory behavior independently in the Asiatic toad

BackgroundIt is well-known that light/dark phase can affect energy expenditure and behaviors of most organisms; however, its influences on individuality (inter-individual variance) and plasticity (intra-individual variance), as well as their associations remain unclear. To approach this question, we repeatedly measured maintenance metabolic rate (MR), exploratory and risk-taking behaviors across light/dark phase four times using wild-caught female Asiatic toads (Bufo gargarizans), and partitioned their variance components with univariate and bivariate mixed-effects models.ResultsThe group means of maintenance MR and risk-taking behavior increased at night, while the group mean of exploratory behavior remained constant throughout the day. At night, the intra-individual variances were elevated in maintenance MR but reduced in exploration, suggesting that phenotypic plasticity was enhanced in the former but constrained in the latter. In addition, maintenance MR was not coupled with exploratory or risk-taking behaviors in daytime or at night, neither at the inter-individual nor intra-individual levels.ConclusionsOur findings suggest that these traits are independently modulated by the light/dark phase, and an allocation energy management model may be applicable in this species. This study sheds new insights into how amphibians adapt nocturnal lifestyle across multiple hierarchy levels via metabolic and behavioral adjustments.

Effects of operational sex ratio and male density on size-dependent mating in Minshan’s toads, Bufo minshanicus, on the Tibetan Plateau of China

Abstract In many animal species, an increase in the operational sex ratio (OSR), density or a combination of both should lead to more intensive competition among individuals of the more abundant sex. To test this, we examined pairing patterns of Minshan’s toad (Bufo minshanicus) from six populations between 2008 and 2015 along the eastern Tibetan Plateau in south-west China. OSRs in breeding aggregations of Minshan’s toad are normally male biased and males actively compete with each other for acquisition and retention of mates. We found evidence that deviations from random mating by size varied between populations and between years according to the magnitude of the OSR and male density. Larger males were generally more successful in pairing than smaller males when the OSR was slightly male biased and male density was high. However, the resulting size-disproportionate mating was more evident when OSR was closer to 1.99, indicating a positive correlation with the intensity of aggressive scramble competition. Thus, the intensity of male-male competition may partly explain variation in size-disproportionate mating among populations.

Identification of peptides of cinobufacini by gel filter chromatography and peptidomics.

Aqueous extract of toad skin (named as Cinobufacini or Huachansu) provides plentiful sources of bioactive peptides that remain undetected and unidentified. High-resolution mass spectrometry-based peptidomics platforms have developed into a major approach to the discovery of natural peptides, with data-dependent acquisition modes providing a wealth of peptide profiling information. In this study, we used a gel- and HLB (a solid phase extraction cartridge)-based two-dimensional separation and purification system and nano-liquid chromatography-tandem mass spectrometry-based peptidomic studies with homology matching for the identification of peptides from Cinobufacini. We evaluated 232 multi-charged peptides and found several specific peptides, some of which were validated by target parallel reaction monitoring mode. These peptides are the first to be identified in Cinobufacini and are completely different from ones identified in toad venom. So, this mapping provides key peptide information for the quality control of Bufo bufo gargarizans skin and its preparation.

Cinobufagin inhibits tumor progression and reduces doxorubicin resistance by enhancing FOXO1-mediated transcription of FCGBP in osteosarcoma

Ethnopharmacological relevanceCinobufagin (Huachansu), an aqueous extract from the dried skin of the toad Bufo bufo gargarizans Cantor (frog skin), is a biologically active ingredient of a traditional Chinese medicine cinobufacini that can treat multiple bone pathological conditions such as bone pain, bone tumors, and osteosarcoma. Aim of the studyThe study aimed to explore the roles and molecular mechanisms of cinobufagin underlying osteosarcoma development and doxorubicin (ADR) resistance. Materials and methodsCell viability, migration, and invasion were examined by CCK-8, wound healing, and Transwell invasion assays, respectively. RNA sequencing analysis was performed in MNNG/HOS cells treated with or without cinobufagin. The relationships of cinobufagin, forkhead box O1 (FOXO1), and Fc fragment of IgG binding protein (FCGBP) were examined by luciferase reporter, immunofluorescence (IF), RT-qPCR, and chromatin immunoprecipitation (ChIP) assays together with weighted gene co-expression network analysis (WGCNA) analysis. Epithelial-mesenchymal transition (EMT) marker levels were examined through the Western blot assay. The function and molecular basis of cinobufagin in osteosarcoma were further investigated by mouse xenograft experiments. ResultsCinobufagin reduced cell viability, weakened ADR resistance, and inhibited cell migration/invasion/EMT in osteosarcoma cells. Cinobufagin enhanced FOXO1-mediated transcription of downstream genes including FCGBP. FCGBP knockdown partly abrogated the effect of cinobufagin on osteosarcoma cell development. Cinobufagin inhibited the growth of mouse osteosarcoma xenografts in vivo. Cinobufagin reduced the expression of Ki-67 and MMP9 and facilitated caspase-3 expression in osteosarcoma xenografts. ConclusionCinobufagin suppressed tumor progression and reduced ADR resistance by potentiating FOXO1-mediated transcription of FCGBP in osteosarcoma.

An integrated strategy to delineate the chemical and dynamic metabolic profile of Huachansu tablets in rat plasma based on UPLC-ESI-QTOF/MSE

Metabolic research is a key method to understand the fate of traditional Chinese medicine (TCM) prescription in vivo and help to explain its pharmacological effects. Huachansu (HCS) tablets are prepared from the dried skin of Bufo gargarizans (Cantor, 1842), which have the effects of detumescence and analgesia, and used in the treatment of middle and advanced tumors. However, an in-depth understanding of the chemical components of HCS tablet and its metabolism in vivo is lacking. In this study, an integrated analytical strategy based on UPLC-ESI-QTOF/MSE was developed to efficiently identify the chemical components, the absorbed prototype compounds and metabolites of HCS tablets given by gavage in rats, and track their dynamic changes. As a result, 77 chemical components of HCS tablets were characterized, including 46 bufadienolides, 11 alkaloids, 10 amino acids and organic acids, 9 nucleosides, and 1 other component. Further more, 16 prototype compounds and 47 metabolites (38 bufadienolides-related and 9 alkaloids-related) were identified in rat plasma. Through the dynamic detection of prototype compounds and metabolites of HCS tablets in vivo, the phenomenon that 3 prototype compounds and 16 metabolites reappeared after a period of disappearance from the plasma was found. Additionally, 8 prototype compounds with large intensity in HCS tablet samples were not detected in the plasma samples of rats given HCS tablets but had metabolites within 0–24 h after administration, indicating that these components were rapidly absorbed and metabolized into metabolites in vivo. It was also observed that 9 metabolites can be driven from various prototype compounds and reach high concentrations in a short time. This study comprehensively identified the xenobiotics and metabolites of HCS tablets in rat plasma sample, systematically discussed their dynamic metabolic process in vivo, which laid a foundation for studying the pharmacodynamic substances of HCS tablets.

Distribution characteristics of Bufo bufo gargarizans resources in China based on quality evaluation of Bufonis Venenum

On the premise of strictly controlling the harvesting conditions of Bufonis Venenum, we studied the relationship between the quality and resource distribution of Bufonis Venenum in China, aiming to provide the data for comprehensively understanding the geographical distribution and characteristics of Bufonis Venenum in China. In this study, 105 samples of Bufonis Venenum were collected from 42 counties and cities in 19 provinces in China, and the material basis and index components were determined by mass spectrometry and high performance liquid chromatography. The obtained data formed the quality database of Bufonis Venenum from different producing areas in China. The analysis of the material basis showed that Bufonis Venenum was mainly produced in two characteristic regions(north area and south area) divided by Qinling Mountains, northern edge of Huaiyang hills and the connecting area of Huang-Huai Plain, Huangshan Mountains, and Tianmu Mountains. Eight differential components were identified in the Bufonis Venenum samples from the south area and the north area. All the Bufonis Venenum samples from the north area showed the content of index components above the requirements of Chinese Pharmacopoeia(2020 edition), while those from the south area had the content of index components lower than the standards of Chinese Pharmacopoeia(2020 edition). The quality evaluation showed uneven distribution of Bufonis Venenum quality, which was high in the north and low in the south. The results provided a research basis for the breeding base selection of Bufo bufo gargarizans.

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.

Cinobufagin inhibits proliferation of acute myeloid leukaemia cells by repressing c-Myc pathway-associated genes

Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na+/K+ ATPase and have similar effects as cardiac glycosides, such as digitoxin or ouabain derived from toxic herbs. Recently, the anti-cancer effects of bufanolides have gained attention, however the underlying molecular mechanisms remain unclear. Selecting cinobufagin as a candidate anti-leukaemia agent, we here conducted transcriptomic analyses on the effect of cinobufagin on human acute myeloid leukaemia (AML) cell lines, HL60 and Kasumi-1. Flow cytometry analysis showed that cinobufagin induced apoptosis in both cell lines. RNA-sequencing (RNA-seq) of the two cell lines treated with cinobufagin revealed commonly downregulated genes with enrichment in the term “Myc active pathway” according to Gene Ontology (GO) analysis. Gene Set Enrichment Analysis (GSEA) of genes downregulated by cinobufagin also showed “MYC_TARGETS_V2” with the highest normalised enrichment score (NES) in both cell lines. In contrast, hallmarks such as “TNFA_SIGNALING_VIA_NFKB”, “APOPTOSIS”, and “TGF_BETA_SIGNALING” were significantly enriched as upregulated gene sets. Epigenetic analysis using chromatin immunoprecipitation and sequencing (ChIP-seq) confirmed that genes encoding cell death-related signalling molecules were upregulated by gain of H3K27ac, whereas downregulation of c-Myc-related genes was not accompanied by H3K27ac alteration. Cinobufagin is an anti-proliferative natural compound with c-Myc-inhibiting and epigenetic-modulating activity in acute myeloid leukaemia.

Effects of Separate and Combined Exposure of Cadmium and Lead on the Endochondral Ossification in Bufo gargarizans.

Cadmium (Cd) and lead (Pb) are ubiquitous in aquatic environments and most studies have examined the potential effects of Cd or Pb alone on aquatic organisms. In the present study, chronic effects of Cd and Pb, alone and in combination, on Bufo gargarizans were investigated by exposing embryos to these contaminants throughout metamorphosis. Significant reductions in body mass and snout-to-vent length were observed in B. gargarizans at Gosner stage 42 (Gs 42) and Gs 46 exposed to a Cd/Pb mixture. Single and combined exposure with Cd and Pb induced histological alterations of the thyroid gland characterized by reduced colloid area and thickness of epithelial cells. There was a significant decrease in the maximum jump distance of froglets exposed to Cd alone and the Cd/Pb mixture, and the jumping capacity showed a positive correlation with hind limb length and tibia/fibula. Moreover, single metals and their mixture induced reduction of endochondral bone formation in B. gargarizans. Transcriptomic and real-time quantitative polymerase chain reaction results showed that genes involved in skeletal ossification (TRα, TRβ, Dio2, Dio3, MMP9, MMP13, Runx1, Runx2, and Runx3) were transcriptionally dysregulated by Cd and Pb exposure alone or in combination. Our results suggested that despite the low concentration tested, the Cd/Pb mixture induced more severe impacts on B. gargarizans. In addition, the Cd/Pb mixture might reduce chances of survival for B. gargarizans froglets by decreasing size at metamorphosis, impaired skeletal ossification, and reduction in jumping ability, which might result from dysregulation of genes involved in thyroid hormone action and endochondral ossification. The findings obtained could add a new dimension to understanding of the mechanisms underpinning skeletal ossification response to heavy metals in amphibians. Environ Toxicol Chem 2022;41:1228-1245. © 2022 SETAC.

Transcriptome Analysis and Identification of the Cholesterol Side Chain Cleavage Enzyme BbgCYP11A1 From Bufo bufo gargarizans.

Bufo bufo gargarizans Cantor are precious medicinal animals in traditional Chinese medicine (TCM). Bufadienolides as the major pharmacological components are generated from the venomous glands of B. bufo gargarizans. Bufadienolides are one type of cardiac aglycone with a six-member lactone ring and have properties of antitumor, cardiotonic, tonsillitis, and anti-inflammatory. The biosynthesis of bufadienolides is complex and unclear. This study explored the transcriptome of three different tissues (skin glands, venom glands, and muscles) of B. bufo gargarizans by high-throughput sequencing. According to the gene tissue–specific expression profile, 389 candidate genes were predicted possibly participating in the bufadienolides biosynthesis pathway. Then, BbgCYP11A1 was identified as a cholesterol side chain cleaving the enzyme in engineering yeast producing cholesterol. Furthermore, the catalytic activity of BbgCYP11A1 was studied with various redox partners. Interestingly, a plant NADPH-cytochrome P450 reductase (CPR) from Anemarrhena asphodeloides showed notably higher production than BbgAdx-2A-BbgAdR from B. bufo gargarizans. These results will provide certainly molecular research to reveal the bufadienolides biosynthesis pathway in B. bufo gargarizans.

Cinobufacini Injection Inhibits the Proliferation of Triple-Negative Breast Cancer Through the Pin1-TAZ Signaling Pathway.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC), which is characterized by the total absence of human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER) expression. Cinobufacini injection (CI) is the aqueous extract from the dry skin of Bufo gargarizans, which is broadly used for the treatment of malignant tumors. However, the potential mechanism of CI against TNBC has not been fully revealed. In this study, we found that CI inhibited the proliferation of MDA-MB-231 and 4T1 cells in a time- and dose-dependent manner. RNA-seq data showed that downregulated and upregulated genes were mainly enriched in biological processes related to tumor cell proliferation, including cell cycle arrest and regulation of apoptosis signaling pathways. Indeed, after CI treatment, the protein level of CDK1 and Bcl-2/Bax decreased, indicating that CI induced the cell cycle of MDA-MB-231 arrest in the G2/M phase and increased the rate of apoptosis. Meanwhile, CI significantly inhibited the growth of tumor in vivo, and RNA-seq data showed that the TAZ signaling pathway played a vital role after CI treatment. Both immunohistochemistry and Western blot analysis confirmed the downregulation of Pin1 and TAZ, caused by CI treatment. Furthermore, the bioinformatics analysis indicated that Pin1 and TAZ were indeed elevated in TNBC patients, with poor staging, classification, and patient survival rate. In conclusion, CI effectively inhibited the proliferation of TNBC in vitro and in vivo and induced their apoptosis and cycle arrest through the Pin1–TAZ pathway.

Effects of nitrite exposure on metamorphosis and skeletal development of Bufo gargarizans.

Nitrite, as a part of nitrogen cycle, is one of the most common toxic compounds in aquatic ecosystems. Since skeletal development is an essential process during amphibian metamorphosis, exposure of larval amphibians to nitrite might disrupt skeletal development. To evaluate whether nitrite affects skeletal development of amphibian larvae, Bufo gargarizans larvae at Gs26 were exposed to 10, 100, 500 and 1000μg/L nitrite-nitrogen (NO2-N) in the present study. The metamorphosis rate, body weight, body length, forelimb length and hindlimb length of B. gargarizans exposed to NO2-N were decreased. The microscopic structures of thyroid gland were altered under NO2-N exposure at Gs42. The skeletal lengths of the humerus, femur and fibulare of tadpole at Gs42 were significantly reduced under 100, 500 and 1000μg/L NO2-N treatment groups, and the lengths of humerus, tibia-fibula and tibiale of tadpole at Gs46 were significantly reduced under 1000μg/L NO2-N treatment groups. In addition, the expression levels of thyroid hormone (TH) and endochondral ossification-related genes of tadpoles at Gs42 and Gs46 were tested by qRT-PCR. Overall, NO2-N exposure could affect the expressions of these genes and then may influence the activity and function of thyroid gland, further disturbing the amphibian metamorphosis and skeletal development of amphibian larvae.

Comparative transcriptomes of three different skin sites for the Asiatic toad (Bufo gargarizans).

Toads release toxic dry secretions from glands in their skin. Toxin possesses a wide range of biological effects, but little is known about its specific gene expression pattern and regulatory mechanisms. The Asiatic toad (Bufo gargarizans) is widely used to produce toxin. Here, we explored the gene expression of 30 tissue samples from three different skin sites (parotoid gland, dorsal skin, and abdomen skin) of B. gargarizans. After de novo assembly, 783,130 unigenes with an average length of 489 bp (N50 = 556 bp) were obtained. A total of 9,248 significant differentially expressed genes (DEGs) were detected. There were 8,819 DEGs between the parotoid gland and abdomen skin and 1,299 DEGs between the dorsal skin and abdomen skin, while only 1,283 DEGs were obtained between the parotoid gland and dorsal skin. Through enrichment analysis, it was found that the detected differential gene expressions corresponded to the different functions of different skin sites. Our key findings were the genetic expression of toxin secretion, the protection function of skin, and the related genes such as HSD3B, Cyp2c, and CAT, LGALS9. In conclusion, we provide useful transcript resources to study the gene expression and gene function of B. gargarizans and other amphibians. The detected DEGs between different sites of the skin provided better insights into the genetic mechanisms of toxin secretion and the protection function of skin for amphibians.

Cinobufagin induces FOXO1-regulated apoptosis, proliferation, migration, and invasion by inhibiting G9a in non-small-cell lung cancer A549 cells

Ethnopharmacological relevanceBufonis (VB), an animal drug called Chansu in China, is the product of the secretion of Bufo gargarizans Cantor or B. melanostictus Schneider. As a traditional Chinese medicine (TCM) for a long time, it has been widely used in the treatment of heart failure, ulcer, pain, and various cancers. Cinobufaginn (CNB), the cardiotonic steroid or bufalene lactone extracted from VB, has the effects of detoxification, detumescence, and analgesia. Aim of the studyThe present study aimed to define the effects of CNB on non-small-cell lung cancer (NSCLC) and identify the potential molecular mechanisms. Materials and methodsA549 cells were treated with cinobufagin and cell viability, apoptosis, migration, and invasion were then evaluated using Cell Counting Kit-8 (CCK8) assays, flow cytometry, and Transwell assays, respectively. Moreover, the levels of proliferating cell nuclear antigen (PCNA), cytokeratin8 (CK8), poly ADP-ribose polymerase (PARP), Caspase3, Caspase8, B-cell lymphoma/lewkmia-2(Bcl-2), Bcl2-Associated X(Bax), forkhead box O1 (FOXO1), and euchromatic histone-lysine N-methyltransferase2 (G9a, EHMT2) in A549 cells were evaluated using qRT–PCR and/or Western blot analysis (WB), Co-IP, immunofluorescence, and immunohistochemistry. An in vivo imaging system, TUNEL, Immunofluorescence, and immunohistochemistry were also used to detect proliferating cell nuclear antigen(PCNA), Ki67, E-Cadherin(E-Cad), FOXO1, and G9a in mouse xenograft model experiments. ResultsCNB suppressed cell proliferation, migration, and invasion but promoted apoptosis in A549 cells in a dose- and time-dependent manner, while cinobufagin had no cytotoxic effect on BEAS-2B cells. In vivo, cinobufagin inhibited the proliferation, migration, and invasion of A549 cells and promoted their apoptosis. The occurrence of the above phenomena was accompanied by an increase in FOXO1 expression and a decrease in G9a expression. In A549 cells, CNB did not reverse the changes in the proliferation, migration, invasion, and apoptosis of A549 cells after FOXO1 was successfully silenced. ConclusionOur study provides the first evidence that cinobufagin suppresses the malignant biological behaviours of NSCLC cells in vivo and in vitro and suggests that mechanistically, this effect may be achieved by inhibiting the expression of the histone methyltransferase G9a and activating the tumour suppressor gene FOXO1. Taken together, our findings provide important insights into the molecular mechanism underlying cinobufagin's anticancer activity, and suggest that cinobufagin could be a candidate for targeted cancer therapy.

北京市两栖爬行动物空间分布格局及影响因素

PDF HTML阅读 XML下载 导出引用 引用提醒 北京市两栖爬行动物空间分布格局及影响因素 DOI: 10.5846/stxb202102260534 作者: 作者单位: 作者简介: 通讯作者: 中图分类号: 基金项目: 生态环境部生物多样性调查与评估项目 Spatial distribution pattern and influencing factors of amphibians and reptiles in Beijing Author: Affiliation: Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:基于遥感和野外调查数据,研究北京市两栖爬行动物空间分布格局及其影响因素。结果显示,北京记录两栖爬行动物2纲3目10科17属22种,其中,保护物种15种,占物种总数的68.19%。物种分布整体呈现由西北向东南递减的空间特征,西北部山区丰富度和相对多度较大,门头沟和延庆物种丰富度最大(14种),占调查物种总数的66.67%,优势种为中国林蛙、黑斑侧褶蛙、中华蟾蜍和山地麻蜥;门头沟、怀柔、房山分布了全市71.54%的种群数量;200-500 m海拔段物种丰富度最高(18种),主要生境为森林灌丛;物种数在6种以上的网格有11个,包括大滩、怀九河、百花山、拒马河、密云水库等。总体来看,物种种类和数量相较之前呈现减少趋势,而土地用途转换具有重要影响。1980-2018年,建设用地表现出以东西城为核心向外围扩张的趋势,侵占的农田占生态系统总转换面积的42.85%;城六区和平原区的主要物种为黑斑侧褶蛙和中华蟾蜍,少有中国林蛙分布。进一步分析两栖动物1500 m的迁徙距离内,森林灌丛和农田为主要的生境类型,城市扩张侵占了部分农田;同时,污染物的排放引起水体水质下降,特别是北运河水系,造成两栖动物的繁殖率降低和栖息地破坏。目前,北京市各类保护地虽覆盖两栖爬行动物种类的90.91%和数量的60.45%,但在怀柔宝山镇白河上段、密云水库来水的潮河上段辛庄桥、平谷洳河大兴庄镇-峪口镇等地,仍然存在较大的保护空缺。对此,建议开展长期定位观测,构建生物多样性观测网络,并探索建立高强度土地利用下的生物多样性保护机制,从而实现生物资源可持续利用。 Abstract:Based on remote sensing and field survey data, the spatial distribution pattern and influencing factors of amphibians and reptiles in Beijing were studied. The results showed that 22 species of amphibians and reptiles were recorded, belonging to 17 genera, 10 families, 3 orders, 2 classes. Among them, there were 15 protected species, accounting for 68.19% of the total species. The spatial distribution of amphibians and reptiles in Beijing decreased from northwest to southeast, and the Northwest Mountainous Areas had higher species richness and relative abundance. The highest species richness (14 species) was in Mentougou District and Yanqing District, accounting for 66.67% of the total species, in which the dominant species were R. chensinensis, P. nigromaculatus, B. gargarizans, and E. brenchleyi. The population in Mentougou, Huairou and Fangshan accounted of 71.54% of Beijing. The highest richness (18 species) was at the altitude 200-500 m, and which main habitat was forest shrub. There were 11 grids with more than 6 species, including Datan, Huaijiu Lake, Baihua Mountain, Juma Lake, Miyun Reservoir, and so on. In general, the species richness and its population number showed a decreasing trend, and the most important impact was land use conversion. During 1980-2018, the construction land presented the trend of expanding to the periphery with the core of Dongcheng District and Xicheng District. The occupied farmland accounts for 42.85% of the total conversion area of the ecosystem. The main species in the six urban districts and plain area are Pelophylax nigromaculatus and Bufo gargarizans, and Rana chensinensis is few. Within the 1500 m migration distance of amphibians, the main habitat types are forest, shrub and farmland, and some farmland is occupied by urban expansion. Pollutants caused the decline of water quality resulted in habitat destruction and the decline of amphibian breeding rate. All kinds of protected areas in Beijing cover 90.91% of the habitat and 60.45% of population of amphibians and reptiles, but there are still some protection gaps in the upper section of Baihe River in Baoshan Town of Huairou District, Xinzhuangqiao in the upper section of Chaohe River from Miyun Reservoir, Daxingzhuang to yukou town of Ruhe River in Pinggu District. Therefore, it is suggested to carry out long-term positioning observation, build Biodiversity Observation Network, and explore the establishment of biodiversity protection mechanism under high-intensity land use, so as to realize the protection and sustainable utilization of biological resources. 参考文献 相似文献 引证文献

Effects of perchlorate and exogenous T4 exposures on development, metamorphosis and endochondral ossification in Bufo gargarizans larvae.

Several endocrine-disrupting chemicals (EDCs) have been proven to interfere with the physiological function of thyroid hormone (TH), which affected growth and development. However, few studies have investigated the effects of EDCs on TH axis with consequence for skeletal development in amphibians. This study thus examined the potential role of perchlorate and T4 in growth, development and endochondral ossification during metamorphosis of Bufo gargarizans. Our studies showed that NaClO₄ treatment caused weight gain and delayed the developmental stage in B. gargarizans tadpoles, while T4 decreased body size and survival rate, accelerated metamorphic duration and increased the risk of early death. Histological sections suggested that NaClO₄ and T4 treatments caused damages to thyroid tissue, such as decreased thyroid gland size, follicle size, colloid area, the height of follicular epithelial cells and the number of follicles. In addition, the double skeletal staining and RT-qPCR showed that NaClO₄ and T4 treatments inhibited the endochondral ossification by regulating TH synthesis (TRs, Dios) and endochondral ossification-related genes (MMPs, Runxs, VEGFs and VEGFRs) expression levels, which might affect terrestrial locomotion and terrestrial life. Altogether, these thyroid injury and gene expression changes as caused by NaClO₄ and T4 may have an influence on development and endochondral ossification during the metamorphosis of amphibians.

Hibernation with rhythmicity: the circadian clock and hormonal adaptations of the hibernating Asiatic toads (Bufo gargarizans).

Hibernation is one of the fundamental strategies in response to cold environmental temperatures. During hibernation, the endocrine and circadian systems ensure minimal expenditure of energy for survival. The circadian rhythms of key hormones, melatonin (MT), corticosterone (CORT), triiodothyronine (T3 ), and thyroxine (T4 ), and the underlying molecular regulatory mechanisms of hibernation have been well determined in mammals but not in ectotherms. Here, a terrestrial hibernating species, Asiatic toad (Bufo gargarizans), was employed to investigate the plasma CORT, MT, T3 , and T4 ; and the retina, brain, and liver mRNA expression of the core clock genes, including circadian locomotor output cycles kaput (Clock), brain and muscle ARNT-like 1 (Bmal1), cryptochrome (Cry) 1 and 2, and period (Per) 1 and 2, at 7-time points over a 24-h period under acute cold (1 day at 4°C), and hibernation (45 days at 4°C). Our results showed that the circadian rhythms of the core clock genes were rather unaffected by acute cold exposure in the retina, unlike the brain and liver. In contrast, during hibernation, the liver clock genes displayed significant circadian oscillations, while those in the retina and brain stopped ticking. Furthermore, plasma CORT expressed circadian oscillations in both groups, and T3 in acute cold exposure group, whereas T4 and MT did not. Our results reveal that the plasma CORT and the liver sustain rhythmicity when the brain was not, indicating that the liver clock along with the adrenal clock synergistically maintains the metabolic requirements to ensure basic survival in hibernating Asiatic toads.

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.

Phylogeographic study of the Bufo gargarizans species complex, with emphasis on Northeast Asia

ABSTRACT We conduct a phylogeographic and population genetic study of the Asiatic toad (Bufo gargarizans) to understand its evolutionary history, and the influence of geology and climate. A total of 292 individuals from 94 locations were genotyped for two mitochondrial loci (cytb, ND2) and five nuclear introns (Sox9-2, Rho-3, CCNB2-3, UCH-2, and DBI-2), and we performed a suite of phylogenetic, population genetic, and divergence dating analyses. The phylogenetic trees constructed using mitochondrial loci inferred B. gargarizans being divided into two major groups: China mainland and Northeast Asia (Northeast China, Russia, and Korean Peninsula). As with previous studies of this species, we recover population genetic structure not tied to geographic region. Additionally, we discover a new genetic clade restricted to Northeast Asia that points towards the Korean Peninsula being a glacial refugium during the Pleistocene. The weak phylogeographic pattern of B. gargarizans is likely the result of multiple biological, anthropogenic, and historical factors – robust dispersal abilities as a consequence of physiological adaptations, human translocation, geologic activity, and glacial cycles of the Pleistocene. We highlight the complex geologic and climatic history of Northeast Asia and encourage further research to understand its impact on the biodiversity in the region.

Evolution of sexual dimorphism in an endemic toad of the Qinghai–Tibet plateau fails to obey Rensch’s rule

According to Rensch's rule, sexual size dimorphism (SSD) decreases with increasing body size in species where females are larger than males, whereas SSD increases with body size when males are larger than females. In this study, we examined body size from 15 populations of Bufo minshanicus in the Qinghai–Tibet plateau in China. We estimated the age of the individuals from eight populations to evaluate Rensch's rule and possible causes of variation in SSD. After or before correcting male and female body size for age differences, the patterns of SSD failed to obey Rensch's rule or its inverse, even though fecundity selection in toads tends to favor larger female body size. We also found that the degree of SSD was positively correlated with the operational sex ratio in the field across B. minshanicus populations. Thus, sexual and fecundity selection play an equal role driving the evolution of SSD within toads. In addition, sex-specific growth rate and age structure explain part of the variation observed in SSD across populations.