Bufo gargarizans:What’s in a Name?

The taxonomic status of Bufo species in East Asia,particularly those closely related to B.gargarizans,has been controversial for over half a century,and recent molecular studies further fueled the dispute.Whether B.andrewsi,B.bankorensis,B.minshanicus,B.sachalinensis,B.tibetanus,and B.tuberculatus constitute valid species or are geographic or ecological variants of B.gargarizans is at the centre of the discussion.Most of these forms possess morphological diagnostic traits,but those differences are minimal and often inconsistent.Molecular data produced many contradictory patterns.Lack of clear diagnostic characters,different usage or definition of various names,mis-identified specimens,limited amount of data or samples,and frequent inter-specific hybridization may all have contributed to the current controversies.Some contradiction may also result from differences between similarity-based clustering and gene tree-based branching patterns.Nevertheless,a dominant allopatric differentiation pattern within this species complex is apparent,and several regional population groups have been identified.Some of the groups resemble old names(e.g.B.minshanicus,B.tibetanus),while others are new.These groups may represent geographic or ecological variants,incipient species,or valid species,and the current data do not warrant a convincing taxonomic revision.To move forward,a single study with comprehensive sampling and genomic data is needed to establish a solid framework,which will define naturally existing population clusters/lineages,and detect their levels of divergence and approximate geographic boundaries.Morphological and ecological examination will follow to understand their biology,determine their taxonomic status,and identify consistent diagnostic characters if necessary.

Changes of Spontaneous Electrical Activities of Olfactory Bulb in Bufo gargarizans during Postembryonic Development

Objective] The aim of this study was to investigate the electrophysiological characteristics of spontaneous electrical activities of olfactory bulb in Bufo gargarizans during postembryonic development. [Method] Microelectrode technique was used to record extracellular spontaneous electrical activities of olfactory bulb in B. gar-garizans. [Result] Along with the development of olfactory bulb, the discharge types increased gradual y. Single spontaneous firing enhanced gradual y and it reached the maximum in the metamorphosis phase. The intensity of consecutive single sponta-neous firing changed slightly at the middle of post-embryonic development, but it was stil higher than in the early phase and the adult phase. Consecutive burst spontaneous firing frequency went down over time. [Conclusion] Along with the de-velopment of olfactory bulb, the neuronal excitability increases step by step and the neuronal electrical activities diversify gradualy.

草甘膦胁迫对中华大蟾蜍(Bufo gargarizans)神经冲动产生和传导的影响

应用电生理方法研究了除草剂草甘膦对中华大蟾蜍（Bufo gargarizans Cantor）坐骨神经干冲动产生和传导的影响。用不同浓度的草甘膦溶液对中华大蟾蜍进行胁迫处理,草甘膦有效成分经由皮肤进入蟾蜍体内而作用于神经系统,利用生物信号采集处理系统测定草甘膦胁迫下中华大蟾蜍离体坐骨神经干的应激反应时间、动作电位幅度和冲动传导速度,结果表明：随着草甘膦溶液浓度的升高,中华大蟾蜍坐骨神经干接受刺激后产生冲动所需的时间逐渐延长,动作电位峰值降低,神经冲动传导速度亦逐渐减慢。草甘膦施用后,中华大蟾蜍7d内的平均应激反应时间与草甘膦浓度呈正相关,而动作电位幅度及传导速度均与草甘膦浓度呈负相关。草甘膦溶液浓度达到推荐农田使用浓度1.64~2.87ml/L时,各处理组蟾蜍的应激反应时间、动作电位幅度和冲动传导速度均与对照组差异极显著（P〈0.01）。同时,随着试验处理时间的延长,中华大蟾蜍神经干对刺激的反应变得更为迟钝,神经冲动的传导速度也进一步减慢。回归分析可知,中华大蟾蜍坐骨神经干的应激反应时间与草甘膦施用后天数呈正相关,而神经传导速度与药后天数呈负相关。由此可以说明,草甘膦胁迫条件下,中华大蟾蜍神经细胞对刺激反应的灵敏性降低，动作电位的产生及传导受到一定程度的抑制和阻碍。

cDNA Cloning and Expression Analysis of Mest Gene in the Bufo gargarizans

The Mest （mesoderm-specific transcript） gene has been considered an imprinting gene in human and mouse, and was also confirmed in other mammals and flowering plants. To investigate the function and evolution of this gene, the cDNA of full length Mest gene was obtained using 5＇- and 3＇-RACE from the Chinese Large Toad （Bufo gargarizans）. The transcript is 1 325bp in length which contains a complete open reading frame （ORF） encoding a polypeptide of 326 amino acids （GenBank accession number： ABQ10905）. There is a typical 0./13 hydrolase fold domain in the putative gene product, and it shows high similarity to sequence of homologous protein of Xenopus tropicali （86%）, mammlian （70% - 80%）. RT-PCR （reverse transcriptase-polymerase chain reaction） analysis demonstrated that the Bufo gargarizans Mest （BgMest） gene is expressed widely in testis, ovary, liver, kidney, spleen, brain, stomach and lung. The conservation of the BgMest gene sequences, protein secondary structure of the BgMest protein, in addition to the expression pattern of the BgMest gene, suggested that the function of BgMest was conserved in amphibians. However, the phylogenetic tree of the imprinting gene of the mammals and other vertebrates examined in this study indicated their divergent origins.

Postembryonic Development of Electrophysiological Characteristic of Primordial Hippocampus in Bufo bufo gargarizans

[Objective] The aim was to probe into developing changes of spontaneous electrical activities of primordial hippocampus in Bufo bufo gargarizans in postembryonic phase. [Method] Microelectrode technique was used to record extracellularly spontaneous electrical activities of neurons in primordial hippocampus of B.bufo gargarizans in postembryonic phase. [Result] Single, consecutive single, burst, consecutive burst and anomalous spontaneous firing were recorded in primordial hippocampus, in which four kinds of spontaneous firings were recorded in forepart of postembryonic development except the anomalous spontaneous firing, and the burst spontaneous firing and consecutive burst spontaneous firing were predominant. The five kinds of spontaneous firings mentioned were recorded in anaphase of postembryonic development and the single and burst spontaneous firing were predominant.Following with the development of primordial hippocampus, the amplitudes of action potential of single firing and consecutive firing enhanced gradually, and it reaches the maximum in the metamorphosis phase; the differences of the frequencies of consecutive single firing and consecutive burst firing were not significant. [Conclusion] Following with the development of primordial hippocampus, the neuronal electrical activities showed diversification, the developmental degree of the primordial hippocampus in the metamorphosis phase was close to the subadult and adult.

Spontaneous firing in olfactory bulb neurons of Bufo bufo gargarizans in and after hibernation

Microelectrode technique was used to record the spontaneous electrical activities of the neurons in olfactory bulb of the Bufo bufo gargarizans, both in hibernation and after hibemation. This study investigated the electrophysiological characteristics of amphibian olfactory bulb in the period of hibernation and after hibernation and its effects on the start of hibernation and spontaneous awakening. The research showed four forms of spontaneous firings： single spontaneous firing, burst spontaneous firing, irregular spontaneous firing and consecutive single spontaneous firing. The single spontaneous firing includes slow depolarized spontaneous firing and fast depolarized spontaneous firing, and the slow depolarized spontaneous firing occurs only during the hibernation period. In hibernation, the low amplitude and low frequency firing with a longer duration may be relevant to maintaining the tonicity of the central nervous system in toads that are in hibernation, and this kind of firing may also provide an excited basis for their arousal from hibernation. After hibernation, the amplitude and frequency of firing increase, but the firing duration gets shorter. This form of short-term firing, which may be a phenomenon of sensory neurons fast adapting, is one of the neuronal mechanisms for the arousal of hibernating animals.

Sexual Dimorphism in the Hindlimb Muscles of the Asiatic Toad(Bufo gargarizans)in Relation to Male Reproductive Success

In many anurans, the forelimb muscles of males are used to grasp females and are often heavier than those of females despite the larger female body size. Such sexual dimorphism in forelimb musculature is thought to result from sexual selection. In addition, the hindlimbs of frogs and toads play an important role in the reproductive process as amplectant males can expel rivals with robust hindlimbs through kicking. In this study, the sexual dimorphism in dry mass for six hindlimb muscles of the Asiatic toad （Bufo gargarizans） was investigated. The results showed that, when controlled for body size, the hindlimb muscle mass of males significantly exceeded that of females for every muscle. The hindlimb muscle mass of amplectant males was also significantly larger than that of non-amplectant males. These results suggested that if strong hindlimb muscles could improve mating success of males, sexual selection would promote the evolution of dimorphism in this character.

中华大蟾蜍(Bufo bufo gargarizans)早期胚胎发育的初步观察

中华大蟾蜍早期胚胎发育分为 2 5个时期 ,在常温下 (1 5 - 1 8℃ )全过程历时 393 .37小时。同时对早期胚胎发育的特点进行了讨论 。

中华大蟾蜍(Bufo bufo gargarizans Contor)早期胚胎发育中RNA的合成

以~3H-尿嘧啶核苷作为RNA合成的标记前身物,用放射自显术研究了中华大蟾蜍早期胚胎发育中RNA合成的速率及其与发育之间的关系。结果表明:从受精卵开始至原肠胚期,RNA的合成活性逐渐增强,神经胚期略有下降;RNA合成速率有明显的动—植物极梯度;在诱导与被诱导部位,RNA合成速率明显高于其它部位.

中华蟾蜍指名亚种(Bufo gargarizan gargarizan)早期胚胎发育

2006年1—2月份对中华蟾蜍指名亚种的早期胚胎发育和产卵行为进行观察.结果表明,蟾蜍整个产卵时间为6.18±0.73 h(N=4),其中雌性产卵8.00±1.20次,雄性射精9.25±1.31次.产卵中,雌性产卵的时间间隔为0.75±0.06 h(N=39),抱对的蟾蜍要游到水面上呼吸,两次呼吸间隔为1.39±0.23 h(N=11).雌蟾蜍最后一次产卵到雌雄分开所需时间为0.96±0.19 h(N=7).从受精卵期至鳃盖完成期,胚胎发育可分为23个时期,在6℃～10℃的水温条件下,历时1111.23±21.64 h(N=7).

Phylogenetic,Demographic and Dating Analyses of Bufo gargarizans Populations from the Zhoushan Archipelago and China's Mainland

Relatively little is known about the relationship between Bufo gargarizans populations from Zhoushan Archipelago and nearby continental regions on the Pacific coast of eastern China.In this paper,155 new specimens of B.gargarizans from Zhoushan Archipelago and adjacent continents and 71 published specimens of B.gargarizans from China's Mainland were studied.Phylogeographical and dating analyses of B.gargarizans were performed using mitochondrial DNA sequencing with a length of 1436 bp.A mt DNA tree that indicated seven major clades was obtained.The earliest split in the mt DNA tree corresponding to the divergence of populations from the western highland region occurred approximately 4.0 million years ago（mya）.A subsequent clade occurred about 3.4 mya,with cladogenesis continuing toward the end of the Pleistocene.The continental clades were distributed in the western,central and northeastern regions of China.Zhoushan Archipelago clades consisted of two largely geographically overlapping subclades with the mt DNA divergence time of 0.73 mya.These results indicated there was extensive dispersal after vicariance.The B.gargarizans populations on Zhoushan Archipelago most probably originated from populations in nearby eastern continental regions of China.It was concluded that geological uplifting during the Pliocene and several sea-level changes in Pleistocene might have influenced the divergence and population demographical history of this species.

Keratin expression during early embryonic development of Bufo bufo gargarizans

Three anti-keratin MAbs were used to identifykeratins expressed in early embryos of Bufo bufogargarizans.MAb AF5 recognized three polypeptides ofkeratin in oocytes,fertilized eggs,up to neurula withMr of 68,65 and 60Kd respectively.At tailbud stage,three other keratins(62,58 and 54Kd)began to expressand could be detected by AF5.MAbs D10 and K12 gavedifferent results,both of them could identify four keratin-like molecules with unusual molecular weights(Mr 98,95,30 and 27 Kd).Moreover,D10 could also detect a 54 Kdkeratin in neurula and tailbud stage embryos,while K12could reveal,beside 54Kd keratin,other four more kera-tins(68,65,62 and 60 Kd).The possible interpretation ofthese results and their implications are discussed.

中华大蟾蜍(Bufo bufo gargarizans)孵化腺细胞的研究

中华大蟾蜍胚胎在尾茅期孵化,其孵化腺细胞(HGC)是从神经管期的表皮外胚层中分化的,呈大瓶状,主要分布于头的前额部和背中线,少量分布于躯干部的背中线。电镜观察表明,神经管期 HGC 通常是几个相连一起,游离面有许多微绒毛,细胞内顶区有大量分泌颗粒,次顶区有许多色素颗粒、粗面内质网和高尔基体;分泌颗粒是由粗面内质网合成,高尔基体浓缩加工而成。随孵化酶的分泌,HGC 表面逐渐缩小,而微绒毛增长和增密。直至变态前退化的 HGC 尚未完全消失。

New cytotoxic C-3 dehydrated bufadienolides from the venom of Bufo bufo gargarizans

Three new C-3 dehydrated bufadienolides were isolated from the venom of Bufo bufo gargarizans. Their structures were elucidated as 5β,12β-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide （1）, 5β,12β- 12,14-dihydroxy-11 -oxobufa-2,20,22-trienolide （2）, and 5β,12β-12,14-dihydroxy-11 -oxobufa-2,20,22- trienolide （3） on the basis of extensive spectroscopic analysis, especially 1D NMR and 2D NMR data. In addition, all three compounds were tested for their cytotoxic activities against A549 and HepG2 cancer cell lines. Compounds 2 and 3 showed significant cytotoxicities with IC50 values less than 10 μmol/L on both cancer cells.

Two new bufotoxins from the skin of Bufo bufo gargarizans

Two new bufotoxins,tolocinobufagin-3-succinoylarginine ester and tolocinobufagin-3-hemisuberate, were isolated from the skin of Bufo bufo gargarizans Cantor.Cinobufagin-3-hemisuberate was also isolated from the natural source for the first time.Their structures were established using spectroscopic methods.

Morphology and mechanical performance between the skin surface of Rana dybowskii and Bufo gargarizans

Anuran skin is a typical natural biomaterial with multifunctional features.A specific comparison of mechanical performance and morphology related to them was performed in the skin of Rana dybowskii and Bufo gargarizans using the tensile testing technique and morphological equipment.Rana dybowskii's skin has soft smooth surface covered by polygonal epidermal cells,while the Bufo gargarizans species has tough and uneven skin surface due to numerous verrucae structures.The collagen fibre bundles in lower dermis of Bufo gargarizans have wavelike organisation while the bundles of Rana dybowskii show a parallel arrangement.The mean elastic modulus of Rana dybowskii was nine times higher than that of Bufo gargarizans.This study clarified that the arrangement of collagen fibres play an important role in the strength and elasticity of skin material.

Kinematics of Chinese toad Bufo gargarizans

A three-dimensional motion force tester for animal was used to measure the reaction forces of the feet of a Chinese toad (B. gargarizans) against a horizontal solid surface on which it crawled. The motion behavior of the Chinese toad was recorded by a high-speed camera on line. The motor function of every foot was analyzed. It was found that the lateral force represents the control ability of side-to-side locomotion, controls the lateral movement, and maintains a stable state for the sustainable creeping gesture. The forefeet play the assistant role and the hind feet play the main role in driving. The normal force of the forefeet is significantly greater than that of the hind feet, so the forefeet play the main role in supporting the body and the hind feet play the assistant role. The normal force is significantly greater than the lateral force and the driving force as well. There is little change for the friction force and the support angle of the all four feet. The average value of the support angle is 70°–80°. The Chinese toad’s vola friction coefficient of the forefeet is less than that of the hind feet. The above results indicate that the difference in kinematics and the material characteristic of the contact skin of the Chinese toad. The results could be useful to the structure design and gait optimization of some robots.

Mutual mate choice in the Asiatic toad, Bufo gargarizans,exerts stabilizing selection on body size

Sexual selection is critical to both reproduction and evolution.The effects of male–male competition and female choice regarding body size have been examined in a large number of taxa,including toad species.Males and females have different optimal reproductive strategies,achieving breeding advantages in discrepant ways.Further,the relative contributions of intra-and inter-sexual size selection vary among species.Thus,to understand the mechanisms affecting mating success,it is important to consider both male–male and male–female interactions simultaneously and elucidate their interrelationship.In this study,we measured body sizes of all mated and unmated individuals in a population of Asiatic toad(Bufo gargarizans)and counted fertilized eggs of several clutches.Based on correlation and regression techniques,we tested for female choice and intra-sexual competition among males relative to body size gradients,and we compared the relative importance of these two processes for mating success.Our results reveal that male–male competition and female choice simultaneously contribute to sexual selection in toads.Furthermore,both interactions are most intense among smaller toads.The synergistic trends of male–male competition and female choice support the mutual mate choice hypothesis and works to stabilize body size in B.gargarizans.Normal distributions of breeding success relative to body size were detected for both sexes,suggesting that medium-sized individuals enjoy a reproductive advantage in the population studied.

蟾蜍TNFRSF11b、TNFRSF9基因克隆及生物信息学分析

目的:克隆中华蟾蜍肿瘤坏死因子受体超家族(TNFRSF)BbgTNFRSF11b、BbgTNFRSF9基因的全长序列,并分析其序列特征。方法:根据转录组测序所得的TNFRSF11b、TNFRSF9基因片段设计特异性引物,以中华蟾蜍蟾皮为材料,利用逆转录聚合酶链式反应(PCR)技术获得BbgTNFRSF11b、BbgTNFRSF9基因全长互补脱氧核糖核酸(cDNA)序列,并采用生物信息学手段分析其序列特征,通过实时荧光定量PCR方法检测BbgTNFRSF11b、BbgTNFRSF9基因在中华蟾蜍7种组织/器官中的表达情况。结果:克隆获得蟾蜍BbgTNFRSF11b基因的全长cDNA序列为1233bp,编码410个氨基酸,理论相对分子质量为46.97kDa,等电点为8.64,不存在跨膜区及信号肽,为亲水性蛋白,具有多个磷酸化位点。BbgTNFRSF9基因的全长cDNA序列为829bp,编码247个氨基酸,理论相对分子质量为67.874kDa,理论等电点为5.12,存在信号肽,为疏水性蛋白,具有多个磷酸化位点,进化树分析发现BbgTNFRSF11b、BbgTNFRSF9基因与其他动物TNFRSF11b、TNFRSF9基因相似度不高,表明该基因虽有特殊结构域但不同物种间差异较大。组织表达分析显示,BbgTNFRSF11b、BbgTNFRSF9基因在耳后腺中表达显著高于其他部位。结论:成功获得蟾蜍BbgTNFRSF11b、BbgTNFRSF9基因序列,掌握其序列特征,为后续深入研究该蛋白的功能提供参考。

基于指纹图谱结合多元统计分析不同干燥方式蟾蜍肉氨基酸类成分

目的:建立不同干燥方式所得蟾蜍肉氨基酸类成分的指纹图谱及4个氨基酸的含量测定方法,比较不同干燥方式对蟾蜍肉品质及其氨基酸含量的影响。方法:采用异硫氰酸苯酯柱前衍生化-高效液相色谱法,建立热风干燥、冷冻干燥、真空干燥和阴干干燥4种方式所得蟾蜍肉干燥样品的指纹图谱,同时测定4个氨基酸含量,并进行相似度评价;运用SPSS 22.0软件进行聚类分析,运用SIMCA 14.1软件进行主成分分析(PCA)和偏最小二乘法-判别分析(PLS-DA)。结果:不同干燥方式得到的16批蟾蜍肉样品指纹图谱相似度结果均大于0.970,共匹配出10个共有峰,指认出缬氨酸、蛋氨酸、异亮氨酸、亮氨酸4个氨基酸;PCA提取出2个主成分,累积方差贡献率为87.95%;PLS-DA得出异亮氨酸、缬氨酸、亮氨酸、蛋氨酸的变量重要性投影(VIP)值均大于0.7,提示4个氨基酸可作为评价不同干燥方式所得蟾蜍肉品质的关键指标;阴干干燥、热风干燥、冷冻干燥和真空干燥4种方式所得蟾蜍肉氨基酸总质量分数测定值分别为7.92%、4.62%~5.37%、7.89%~9.02%和4.60%~9.24%。结论:所建立的指纹图谱及含量测定方法稳定可行,结合多元统计分析发现不同干燥方式对蟾蜍肉氨基酸含量产生较大影响,可为蟾蜍肉干燥方式和质量控制研究提供参考。