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.

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.

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.

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.

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.

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.

Competitive Strategies of Two Species of Co-occuring Tadpoles

We examined the competitive ability of larval toads （Bufo gargarizans ） and frogs （Rana kukunoris ） which co-occur in natural pools in the Jiuzhaigou Nature Reserve. We measured the activity level, growth rate, mass at metamorphosis and larval period in a laboratory experiment. Tadpoles of B. gargarizans were significantly more active when food was abundant than scarce, while there was no significant difference in the activity of R. kukunoris tadpoles at different food levels. At low food availability, mass at metamorphosis and growth rate of R. kukunoris were significantly increased in the presence of B. gargarizans, whereas the presence of R. kukunoris had no significant effect on the mass and growth rate of B. gargarizans. In all treatments, the larval period of B. gargarizans at low food availability was the shortest. These results suggest that B. gargarizans can adapt to different food levels by changing their activity rate. At high food level, B. gargarizans increased activity to gain more food. At low food level, B. gargarizans decreased activity and achieved early metamorphosis. However, when food resources were limited, R. kukunoris could gain more food than B.gargarizans .

A Robust Noninvasive Approach to Study Gut Microbiota Structure of Amphibian Tadpoles by Feces

The 16S rDNA amplicon high-throughput sequencing technique provides a robust and inexpensive approach to detect the gut microbiota of amphibians. Since different experimental protocols generate technical biases in drawing the gut microbiota profiles, the integrative analysis of gut microbiota produced by different studies must be performed with circumspection. In this study, we compared the efficacy of two DNA extraction methods （i.e., a phenol-chloroform method and TIANamp Stool DNA Kit） in describing intestinal and fecal bacterial communities of transplanted Asiatic toad （Bufo gargarizans） tadpoles. In terms of the DNA extraction quality （i.e., DNA purity and yield rate） and the consistency in between fecal and intestinal microbiota structures （i.e., a and 13 diversity indices）, the phenol-chloroform method was more robust than this commercial stool kit in profiling gut microbiota of tadpoles with feces.

中华蟾蜍资源现状及其保护对策

中华蟾蜍(Bufo gargarizans)资源面临生态环境恶化,数量锐减,市场需求量大,资源短缺的现状。本文对养殖历史和产业现状进行概述,分析了资源保护措施有待加强、产品加工不完善,利用率不高、养殖政策引导扶持力度不足及缺乏养殖加工规范化、标准化技术等存在的问题,并针对性提出加强科学监测,强化宣传保护,科学增殖放流,加大科研投入,进行种质资源及人工繁殖研究,提高蟾蜍及其制品加工利用广度和科技含量及引导合法养殖,扩大养殖产业规模等相应的对策。

Sex But Not Altitude, Modulates Phenotypic Covariations Between Growth and Physiological Traits in Adult Asiatic Toads

The pace-of-life syndrome(POLS) hypothesis predicts that most variation in life history, physiology,and behavior among individuals, popula tions, and species falls along a continuum from slow to fast pace of life. While there is evidence for climatic gradientmediated POLS patterns among species, this approach has rarely been explicitly used to study POLS patterns among-and within-populations. In addition, the roles of sex in POLS evolution among-or within-populations are largely unknown. In this study, we investigated the effects of altitudinal gradient and sex on the covariations between growth rate and several physiological traits closely associated with POLS(blood glucose, baselineand stress-induced glucocorticoids(GCs), hemolysis and hemagglutination) in the Asiatic toad Bufo gargarizans.Contrary to our expectation, altitudinal gradient had no influence on the covariations between growth rate and physiological traits, neither at the among-nor withinpopulation level, indicating that these trait integrations have similar fitness payoffs across hierarchical levels.In contrast, we found evidence for sex-specific POLS composition: there was a negative covariance structure between growth rate and baseline GCs-but only in females, and a positive covariance structure between growth rate and baseline GCs-but only in females, and a positive covariance structure between growth rate and hemagglutination-but only in males. This observation indicates that these trait associations differ dramatically in advancing fitness for each sex, and supports the idea that sex-specific POLS composition could evolve in species in which the reproductive roles largely differ between the sexes.

Male size does not correlate with fertilization success in two bufonid toads that show size-assortative mating

We examined sexual size dimorphism （SSD）, mating pattem, fertilization efficiency and female reproductive traits in two bufonid toads （Bufo gargarizans and Duttaphrynus melanostictus） to test the idea that importance of male body size for egg fertilization success depends on the mating pattern. Female-biased SSD was evident only in D. melanostictus. Female B. gar- garizans laid fewer larger eggs nearly three months earlier than did female D. melanostictus. Fertilization efficieneies on average were higher in B. gargarizans （95%） than in D. melanostictus （91%）. Though differing in the degree of SSD, body size, breeding season, clutch size, egg size and fertilization efficiency, the two toads were similar in four aspects： （1） both showed size-assortative mating; （2） females did not tradeoff egg size against egg number; （3） male size, clutch size and clutch dry mass were greater in male-larger than in female-larger pairs after accounting for female snout-vent length （SVL）; and （4） the ratio of male to female SVL did not affect fertilization efficiency. Our data show that： （1） a female preference for large males is likely not important in terms of egg fertilization success; （2） a male preference for large females is likely important because larger females are more fecund; and （3） size-assortative mating arises from a male preference for large females. Our study demonstrates that male size is not always important for egg fertilization success in anurans that show size-assortative mating.

孕酮诱发蟾蜍卵母细胞成熟作用部位的探讨

冬眠蟾蜍长足卵母胞胞,经手工剥除其卵巢膜、滤泡膜、透明带和质膜,包埋在琼脂块里,孕酮作用3小时(18±1℃)后,该细胞质团块中出现促成熟活性物质(MPF);将含有此MPF的微量卵质(约50毫微升),注入未经激素处理的卵球,能诱发后者恢复减数分裂,胚泡破裂,排出第一极体,正常抵达第二次成熟分裂中期。如果在去除上述卵外和卵表膜层结构的同时,剔除其细胞核(胚泡),然后包裹在琼脂中,经孕酮处理3小时左右,照样能够诱发产生促成熟活性物质;微量细胞质的转移,照样能使未经激素处理的受体卵正常成熟。随着供体卵质块与孕酮接触时间的延长,其诱发受体卵成熟的百分率逐渐增高。孕酮处理后9小时的供体卵质块,几乎全部能使受体卵正常成熟。上述结果表明,在本实验处理的条件下,孕酮诱发中华大蟾蜍卵母细胞形成促成熟活性物质的过程,既不依赖于卵表透明带与质膜,也不依赖于细胞核,而是细胞质自身活动的结果;显然,孕酮诱发蟾蜍卵母细胞成熟的初始作用部位是在细胞质。

Body size variation of four latitudinally-separated populations of a toad species:age and growth rate as the proximate determinants

Age and growth rate are critical parameters determining the body size of adult anurans,which grow indeterminately.Therefore,evaluating their relative importance and selective pressures acting on them may help to explain variation in body size of anurans across geographical populations.Based on the data on body size and skeletochronological age of 4 Asiatic toad(Bufo gargarizans)populations that are widely separated in latitude,the present study showed that toads at the northern extremity of the range were the oldest,on average,and grew relatively fast,toads at the southern end of the range were relatively old but grew fastest,whereas mid-latitude toads were intermediate in both traits.These results indicated that relative contributions of age and growth rate to body size differed among populations.From a life history perspective,greater age attained by toads at higher latitudes should result from delayed maturation,and faster growth from an adaptive response to strong seasonal time constraints;in contrast,selective pressures on the 2 parameters at lower latitudes might be associated,respectively,with low adult death rates and longer favorable growing seasons.