菜阳河自然保护区两栖爬行动物调查报告

："))) 年( * ’ 月对云南省思茅菜阳河省级自然保护区两栖爬行动物多样性进行了调查。调查结果表明：菜 阳河自然保护区共有两栖爬行动物+, 种。其中，两栖类#, 种，隶属于" 目’ 科!+ 属；爬行类() 种，隶属于# 目!- 科$! 属。属于国家!级重点保护的种类有" 种；"级重点保护的种类有( 种；云南省省级保护动物# 种； 列入云南省“三有”动物的!) 种。结合调查结果，对菜阳河自然保护区提出了进行功能区划，分区管理，以及 严格管理农药的使用等两方面的建议。

Molecular phylogeography and population structure of a mid-elevation montane frog Leptobrachium ailaonicum in a fragmented habitat of southwest China

Leptobrachium ailaonicum is a vulnerable anuran restricted to a patchy distribution associated with small mountain streams surrounded by forested slopes at mid-elevations (approximately 2000-2600 m) in the subtropical Mount Wuliang and Mount Ailao ranges in southwest China (Yunnan Province) and northern Vietnam. Given high habitat specificity and lack of suitable habitat in lower elevations between these ranges, we hypothesized limited gene flow between populations throughout its range. We used two mitochondrial genes to construct a phylogeographic pattern within this species in order to test our hypothesis. We also examined whether this phylogeographic pattern is a response to past geological events and/or climatic oscillations. A total of 1989 base pairs were obtained from 81 individuals of nine populations yielding 51 unique haplotypes. Both Bayesian and maximum parsimony phylogenetic analyses revealed four deeply divergent and reciprocally monophyletic mtDNA lineages that approximately correspond to four geographical regions separated by deep river valleys. These results suggest a long history of allopatric separation by vicariance. The distinct geographic distributions of four major clades and the estimated divergence time suggest spatial and temporal separations that coincide with climatic and paleogeographic changes following the orogeny and uplift of Mount Ailao during the late Miocene to mid Pliocene in southwest China. At the southern distribution, the presence of two sympatric yet differentiated clades in two areas are interpreted as a result of secondary contact between previously allopatric populations during cooler Pleistocene glacial cycles. Analysis of molecular variance indicates that most of the observed genetic variation occurs among the four regions implying long-term interruption of maternal gene flow, suggesting that L ailaonicum may represent more than one distinct species and should at least be separated into four management units corresponding to these four geographic lineages for conservation. (C) 2009 Elsevier Inc. All rights reserved.

Molecular Characterization and Functional Commonality of Nucleophosmin/Nucleoplasmin in Two Cyprinid Fish

Nucleophosmin/nucleoplasmin has been studied mostly in mammals and amphibians. To clarify the characteristics and function of nucleophosmin/nucleoplasmin in teleost fish, we cloned a full-length cDNA sequence from two cyprinid fish, Carassius auratus gibelio and Carassius auratus. Molecular characterization and multiple sequence alignments suggested that they are the homologs of nucleophosmin. RT-PCR and Western blot detected a specific expression in gonads, and immunofluorescence localization revealed their distribution in oogenic and spermatogenic cells. Furthermore, a sperm decondensation function was demonstrated by immunodepletion and in vitro sperm decondensation experiments. The data suggest that the cloned nucleophosmin should share expressional and functional characterization with nucleoplasmin and therefore provide novel evidence for a functional commonality of nucleophosmin and nucleoplasmin in fish.

First report of two Balantidium species from the Chinese giant salamander, Andrias davidianus: Balantidium sinensis Nie 1935 and Balantidium andianusis n. sp.

Two ciliated protozoa, Balantidium sinensis Nie 1935 and Balantidium andianusi n. sp., were isolated from the feces of a wild Chinese giant salamander (Andrias davidianus) captured from the mountainous area of Shiyan, Hubei Province, Central China in October 2006. It is the first report of Balantidium species inhabiting Cryptobranchoidea amphibians. The occurrence of B. sinensis in A. davidianus should be a new record because the type specimens were first discovered and named by Nie in 1935 from Rana nigromaculata and Rana plancyi. For the lack of enough descriptions of taxonomic features in the previous report, it was re-described in detail and compared with Nie's type specimens and B. giganteum to complete the morphological descriptions in the present work. B. andianusi n. sp. was considered to be a new species based on its unique morphological characteristics, especially the high length/width ratio of the vestibulum (8:1). Comparisons were also made among Balantidium species that were found from urodele amphibians.

The distribution and economic losses of alien species invasion to China

Invasive alien species have become one of the most serious environmental issues in the world. Data of taxon, origin, pathway, and environmental impacts of invasive alien microorganisms, invertebrates, amphibians and reptiles, fish, birds, mammals, herbs, trees, and, marine organisms in terrestrial, aquatic, and marine ecosystems of China were analyzed during 2001 and 2003, based on literature retrieval and field survey. There were 283 invasive alien species in China, and the number of species of invasive alien microorganisms, aquatic plants, terrestrial plants, aquatic invertebrates, terrestrial invertebrates, amphibians and reptiles, fish, and mammals were 19, 18, 170, 25, 33, 3, 10, and 5, respectively. The proportion of invasive alien species originated from America, Europe, Asia, Africa, and Oceania were 55.1, 21.7, 9.9, 8.1, and 0.6%, respectively. Methods for estimation of direct economic losses to agriculture, forestry, stockbreeding, fishery, road and water transportation, storage, water conservancy, environment and public facilities, and human health were established. Methods for estimation of indirect economic losses caused by invasive alien species to service functions of forest ecosystems, agricultural ecosystems, grassland ecosystems, and wetland ecosystems were also established. The total economic losses caused by invasive alien species to China were to the time of USD 14.45 billion, with direct and indirect economic losses accounting for 16.59% and 83.41% of total economic losses, respectively.

The status and causes of alien species invasion in China

Data of classification, origin, pathway and environmental impacts of invasive alien micro-organisms, invertebrates, amphibians and reptiles, fish, birds, mammals, weeds, trees, and marine organisms in terrestrial, aquatic and marine ecosystems of China, were analyzed, based on literature retrieval, field survey and consultation. Some 283 invasive alien species were recorded in China, including 19 invasive alien micro-organisms, 18 aquatic plants, 170 terrestrial plants, 25 aquatic invertebrates, 33 terrestrial invertebrates, 3 amphibians and reptiles, 10 fish, and 5 mammals. Of the invasive alien species, 55.1% originated from North and South America, 21.7% from Europe, 9.9% from Asia, 8.1% from Africa and 0.6% from Oceania. Many institutions and individuals in China lack adequate knowledge of ecological and environmental consequences caused by invasive alien species, with some ignorance of the dangerous invasion in the introduction of alien species. For instance, 50.0% of invasive alien plants were intentionally introduced as pasture, feedingstuff, ornamental plants, textile plants, medicinal plants, vegetables, or lawn plants, 25% of alien invasive animals were intentionally introduced for cultivation, ornament, or biological control, In addition, more efforts are being made in the introduction of alien species, and little attention is paid on the management of introduced alien species, which may cause their escape into natural environment and potential threats to the environment. There were also gaps in quarantine system in China. All microorganisms were unintentionally introduced, through timber, seedling, flowerpot, or soil; 76.3% of alien invasive animals invaded through commodity or transportation facility because of the failure of quarantine. Therefore, quarantine measures should be strictly implemented; and meanwhile the intentional introduction of alien species should be strictly managed and a system of risk assessment should be implemented.

Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp (Carassius auratus gibelio bloch) as revealed by RAPD assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp.

三种两栖类动物皮肤和脑中活性多肽结构、功能与多样性分析

两栖动物是最原始的陆生脊椎动物，分布比较广泛。无尾目两栖动物现有3000 多种，它们皮肤裸露、光滑，为适应广泛的栖息地和生态条件，已进化出各种有效的皮肤防御系统。抗菌肽（Antimicrobial peptides，AMPs）作为两栖类先天防御系统的重要组成部分，在皮肤分泌液中含量异常丰富。我们以来源于云南省普洱市景东县的铃蟾科微蹼铃蟾（Bombina microdeladigitora）和楚雄州双柏县雨蛙科华西雨蛙（Hyla annectans）为实验材料，对其皮肤分泌液中抗菌肽的分子多样性并对其结构和功能进行研究。微蹼铃蟾皮肤抗菌肽多样性非常丰富，我们从单一个体中克隆得到了64 条编码不同抗菌肽的cDNA 序列，其中有两条序列只编码Maximin 一种抗菌肽，其余 62 条均编码Maximin 和Maximin H 两类抗菌肽。这64 条cDNA 序列共编码44 种Maximins 和30 种Maximin Hs，其中有32 种Maximins 和20 种Maximin Hs 为新鉴定的抗菌肽，其余和铃蟾属其它种中发现的抗菌肽具有相同的序列。除了皮肤外，两栖动物的脑也是抗菌肽的丰富资源库。我们分别从微蹼铃蟾和大蹼铃蟾（B. maxima）脑中得到了大量新的抗菌肽cDNA 序列。其中从微蹼铃蟾脑中克隆到21 条新的cDNA序列，共编码16 种Maximins 和10 种Maximin Hs，其中7 种Maximins 和4 种Maximin Hs 为新鉴定的抗菌肽。从大蹼铃蟾脑中克隆到39 条新的cDNA 序列，编码27 种Maximins 和20 种Maximin Hs，其中16 种 Maximins 和12 种Maximin Hs 为新鉴定的抗菌肽。在以上新鉴定的抗菌肽中，Maximins 均为阳离子抗菌肽，Maximin Hs 中除以前鉴定的Maximin H5 外，尚有十余种阴离子抗菌肽。抗菌肽碱基转换/颠换（R=s/v）分析表明，RMaximin<1 而RMaximin H>1，说明这两种抗菌肽碱基转换和颠换发生的几率并不相同，Maximin 间差异主要由碱基颠换引起，而Maximin H 则主要由碱基转换引起。种间进化分析表明，大蹼铃蟾和微蹼铃蟾的遗传距离较近，而它们与欧洲花铃蟾（B. variegata）的遗传距离均较远。种内各部分遗传距离差异较大。与信号肽和酸性间隔肽相比，成熟肽的遗传距离明显增大，其中Maximin 的进化速度比Maximin H 更快。抗菌肽Maximin 和Maximin H 种内、种间均存在正选择（ω>1），而信号肽和酸性间隔肽在分化过程中没有正选择（ω<1），说明Maximin 和Maximin H 经受着达尔文正选择驱动的快速进化，是抗菌肽多样性产生的根本原因。这与抗菌肽参与最终的生物防御功能，从而增加物种对环境的适应是一致的。功能研究发现，有些微蹼铃蟾Maximins 抗菌肽是多功能分子，不但对革兰氏阴性菌、革兰氏阳性菌和真菌起抗菌作用，而且还具有很强的抗氧化功能。脑中抗菌肽基因的大量表达也预示着抗菌肽可能在神经信号传导中起一定作用。用基因克隆方法我们从微蹼铃蟾皮肤得到大量缓激肽前体序列，由1-4 个拷贝的Bombinakinin 或1-4 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成。这与大蹼铃蟾皮肤中缓激肽前体由1-8 个拷贝的Bombinakinin 或1-8 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成有所不同。按同样方法，我们从大蹼铃蟾脑中也得到了三条缓激肽前体序列，其中两条含有6 个 Bombinakinin 拷贝，另一条含2 个Bombinakinin 拷贝。通过比较只含Bombinakinin 和同时含有Bombinakinin 和Bombinakinin-GAP 的前体cDNA 序列后发现， 前者序列中缺失了一段碱基序列TGCGGGTA， 从而导致移码突变， 终止了 Bombinakinin-GAP 的表达。通过生物化学的手段从微蹼铃蟾皮肤分泌液中分离到一种丝氨酸蛋白酶抑制剂BMSI1，与铃蟾属其它种中的胰蛋白酶抑制剂具有很高的相似性。根据已有铃蟾属丝氨酸蛋白酶抑制剂cDNA 序列设计引物，以皮肤cDNA 为模板，扩增丝氨酸蛋白酶抑制剂的基因序列，结果得到两条不同的序列。这两条前体序列与铃蟾属其它两栖动物皮肤中的丝氨酸蛋白酶抑制剂具有高度相似性（>70%），而且它们都含有10 个半胱氨酸残基。BMSI1 对五种丝氨酸蛋白水解发色底物的抑制活性测定表明，BMSI 1 能抑制胰蛋白酶和凝血酶的水解活性，其K(i)分别为0.02 μM 和0.15 μM。通过随机筛选cDNA 文库的方法，我们从大蹼铃蟾脑中得到了一条完整的 Somatostatin（SST）序列，根据该序列，我们在大蹼铃蟾和微蹼铃蟾脑cDNA 文库中筛选到两条变异体序列SST-L(Leu11-SST-14)和SST-R(Arg14-SST-14)。功能研究表明，这两种变异体具有和SST 相似的生物学功能，可抑制肿瘤细胞增殖、抑制细胞因子释放以及具有一定的镇痛作用。从大蹼铃蟾和微蹼铃蟾脑中得到了阿片肽前体POMC 和Proenkephalin 的 cDNA 序列，序列比对发现与东方铃蟾具有较高的同源性。从华西雨蛙皮肤cDNA 中克隆得到两类活性多肽，命名为Annins。其中一类为抗菌肽类似肽，共11 条序列，编码单一的成熟肽序列，其信号肽与雨蛙科信号肽具有很高的同源性，但酸性间隔肽和成熟肽相差较大。其成熟肽由15-17 个氨基酸残基组成，活性分析表明无抗菌和抗氧化作用，但在较高浓度时对部分细菌和多种细胞有促进生长作用，推测可能在使伤口快速愈合方面起重要作用；另一类编码具有2 个拷贝的成熟肽序列，成熟肽由5 个氨基酸残基组成，具有一定的镇痛活性，其镇痛机理可能是拮抗bradykinin 作用。

若尔盖湿地公路对两栖类的生态影响

本文考察了若尔盖高寒泥炭湿地公路对高原林蛙（Rana kukunoris）、倭蛙（Narorana pleskei）和岷山蟾蜍（Bufo minshanicus）的生态影响。分析了公路对两栖动物空间分布和栖息地利用的影响，并用IBM模型探讨其可能作用机制，考察了两栖动物公路死亡的季节差异及影响公路死亡空间分布的景观因素。最后通过对若尔盖高寒湿地两栖动物陆地核心栖息地的分析，为若尔盖路域栖息地的管理提供依据。 1. 对公路周边6个沼泽水凼群进行了调查，每个样地设置5条样线（距离公路10m、20m、50m、100m和150m）。调查表明，在繁殖季节(5月)，距离公路距离对高原林蛙和倭蛙的相对数量都有显著作用，其效应明显大于其他各项栖息地环境参数。公路导致高原林蛙和倭蛙在公路周边种群密度降低，其相对数量从距离公路100m处到公路边缘一直呈现逐渐降低的趋势。在繁殖季节，若尔盖高寒湿地的公路生态影响域大约在100-150m之间，这一距离远远大于森林栖息地中公路对两栖类的生态影响域（35-40 m）。 在繁殖后期（9月），对公路周边16个草地样点的样线调查表明，公路对周边高原林蛙和倭蛙密度分布并未造成显著影响。 2. 二次模型的拟合表明繁殖季节高原林蛙和倭蛙在公路周边的密度分布符合钟型曲线。前人对森林公路两侧两栖类分布的研究也显示了类似的规律。我们通过基于个体的模型，模拟在了公路边缘100单位距离内的栖息地空间，栖息地环境质量呈梯度变化，动物个体在其中通过随机运动寻找适宜的栖息地。拟合结果表明，动物个体仅仅依照简单的运动规则寻找适宜栖息地，这种活动就可以导致公路周边栖息地中的动物分布曲线出现3个局部峰。公路周边两栖动物的钟型分布曲线可能仅仅是个体寻找适宜栖息地过程中出现的临时性群体分布模式。 3. 在若尔盖高寒湿地，公路交通造成了大量两栖类死亡。但是公路两栖类动物死亡的季节分布很不均匀：5月、8月和9月死亡数量很高，而7月和10月死亡数量却很低。这种季节性差异和两栖类各个生活史阶段的迁移运动有密切的关系。利用景观参数的逻辑斯蒂回归模型显示，距离公路1000-2000m范围内的湿草地比例对三种两栖类公路死亡概率均有很强的贡献。湿草地这一栖息地类型分类中有大量的沼泽水体，是两栖类重要的繁殖点和取食点。两栖类公路死亡概率湿草地的关系从一个侧面表明，要维持一个区域较高的两栖类种群数量，需要1000-2000m半径范围内存在大面积的湿草地。 4. 高原林蛙和岷山蟾蜍不同性别和年龄个体分布点的水体距离存在显著差异。不同种类、年龄的两栖类分布点距离水体距离的差异可能是由于对水体的依赖性造成的。而相同种类、年龄段的个体中，高原林蛙雌性、岷山蟾蜍亚成体和雌性的体重与分布点距水体距离有显著负相关，这可能是因为体重更大的个体对水体的依赖性更弱。考虑到过大的陆地核心栖息地面积在实际保护工作中存在操作上的困难，因此我们认为可以以水体周边90%个体的分布区为低限确定3种两栖类的最小陆地核心栖息地。但是，在同样的水体距离-两栖类密度分布格局下，水体的面积和分形参数对最小陆地核心栖息地半径的确定有一定影响。 Ecological effects of alpine wetland road on Rana kukunoris, Narorana pleskei, Bufo minshanicus was studied in Zoige wetland. The effects of road on distribution of amphibians and its possible underline mechanism was discussed based on empirical data and computer simulation. Road killed amphibians was surveyed in different season and those landscape factor which could have impact on road killing distribution was analyses. Core terrestrial habitat of amphibians in Zoige wetland was discussed in the consideration of conservation management. 1. Six pool groups was investigated in breeding season (May) of R. kukunoris, N. pleskei. Five transects at distance of 10m, 20m, 50m, 100m and 150m from road edge was surveyed in each pool groups. There was a significant effects of distance from road edge on relative counts of R. kukunoris, N. pleskei, which is much important than effects of other environmental factors. Road caused the density of R. kukunoris, N. pleskei decreased from distance of 100m from road to 10m from road. Road ecological effect zone of alpine wetland for amphibians is about 100-150m. It is much wider than those of forest roads, which is about 35-40m. However, studies on 16 grassland near road showed no significant effect of road on amphibians after breeding season (Sep.). 2. Quadratic model fit indicated that the distribution of R. kukunoris and N. Pleskei followed a hump like curve. Previous studies on forest road showed similar results. A 100×100 habitat with gradual environment besides road was simulated with a individual-based model, and animal seek for suitable habitat with stochastic locomotion in it. Simulation results indicated that 3 density peak of animal distribution can emergent followed a simply rules. The hump like density cure could be a temporal swarm pattern during the process of individual seeking for habitat. 3. Road traffic caused mass death of amphibians in Zoige wetland. There was much road killed amphibians in May, Aug and Sep than those in July and Oct. The fluctuation of road kill could be related with migration of amphibians between seasons. Logistic regression of landscape variables indicated that wet grassland in 1000-2000m is essential to predict the probability of road kill. Wet grassland is an important breeding and forage habitat for amphibians. It also indicated that mass wet grassland in 1000-2000m is essential for maintain a big amphibian population. 4. There was significant differences among distance from aquatic site of subadults, female and males of R. kukunoris and B. Minshanicus. Possibly, it was because of their dependence on water. There was a significant negative relationship between distance from aquatic site and individuals body mass. Estimates of core habitat that are too large may make it difficult to establish protective regulations. The smallest suitable terrestrial core habitats were defined as the terrestrial habitats used during migration to and from the wetlands, and for foraging by 90% of any life stage (adults, and subadults) in a season. However, even with the same amphibian distribution pattern along the distance from aquatic sites, the radii of smallest suitable terrestrial core habitats will be varied with the fractal parameters of aquatic site.

Modeling trophic positions of the alpine meadow ecosystem combining stable carbon and nitrogen isotope ratios

Stable carbon and nitrogen isotope ratios of single tissues or whole bodies were analyzed to establish trophic positions of main consumers living at the alpine meadow ecosystem in the Tibetan Plateau. The results demonstrated that delta C-13 and delta N-15 values of vertebrates showed great variations and ranged from -26.83 to -22.51 parts per thousand and from 2.33 to 8.44 parts per thousand, respectively. Plateau pika, root vole, plateau hare, infants of rodents and hatchlings of passerine bird species had the lowest delta C-13 and delta N-15 values. delta C-13 and delta N-15 values of omnivorous and insectivorous birds and amphibians showed intermediate. Carnivorous species, steppe polecat and Upland buzzard, and omnivorous Robin accentor and White wagtail possessed extremely higher VC and delta N-15 values. Omnivorous birds captured in earlier year had significantly less negative delta C-13 and greater delta N-15 values than those captured later. Based on steady angular enrichment between trophic levels, an "alpha and vector model" combing delta C-13 and delta N-15 values was introduced to reveal trophic positions, the results indicated that Tibetan sheep, Tibetan yak, plateau pika, root vole, plateau hare, infants of small rodents showed the lowest trophic positions (TP 1.81-2.38). While omnivorous and insectivorous birds, their hatchlings and amphibians showed intermediate trophic positions (TP 2.06-2.89), carnivorous species steppe polecat and Upland buzzard, migrant birds possessed extremely higher trophic positions (TP 2.89-3.05). The isotopic investigation of organisms and the introduced "alpha and vector model" successfully demonstrated the same trophic positions and diet prediction of consumers as nitrogen enrichment model at the alpine meadow ecosystem. Besides of this information, the "alpha and vector model" can also be incorporated into multiple isotope signatures to infer trophic relationships. This angular enrichment model has the potential to address basic ecological questions, such as trophic structure, trophic dynamics, and energy flow in other terrestrial ecosystems of properly handled. (C) 2005 Elsevier B.V. All rights reserved.

Ras controls melanocyte expansion during zebrafish fin stripe regeneration.

Regenerative medicine for complex tissues like limbs will require the provision or activation of precursors for different cell types, in the correct number, and with the appropriate instructions. These strategies can be guided by what is learned from spectacular events of natural limb or fin regeneration in urodele amphibians and teleost fish. Following zebrafish fin amputation, melanocyte stripes faithfully regenerate in tandem with complex fin structures. Distinct populations of melanocyte precursors emerge and differentiate to pigment regenerating fins, yet the regulation of their proliferation and patterning is incompletely understood. Here, we found that transgenic increases in active Ras dose-dependently hyperpigmented regenerating zebrafish fins. Lineage tracing and marker analysis indicated that increases in active Ras stimulated the in situ amplification of undifferentiated melanocyte precursors expressing mitfa and kita. Active Ras also hyperpigmented early fin regenerates of kita mutants, which are normally devoid of primary regeneration melanocytes, suppressing defects in precursor function and survival. By contrast, this protocol had no noticeable impact on pigmentation by secondary regulatory melanocyte precursors in late-stage kita regenerates. Our results provide evidence that Ras activity levels control the repopulation and expansion of adult melanocyte precursors after tissue loss, enabling the recovery of patterned melanocyte stripes during zebrafish appendage regeneration.

Granular gland transcriptomes in stimulated amphibian skin secretions.

Amphibian defensive skin secretions are complex, species-specific cocktails of biologically active molecules, including many uncharacterized peptides. The study of such secretions for novel peptide discovery is time-limited, as amphibians are in rapid global decline. While secretion proteome analysis is non-lethal, transcriptome analysis has until now required killing of specimens prior to skin dissection for cDNA library construction. Here we present the discovery that polyadenylated mRNAs encoding dermal granular gland peptides are present in defensive skin secretions, stabilized by endogenous nucleic acid-binding amphipathic peptides. Thus parallel secretory proteome and transcriptome analyses can be performed without killing the specimen in this model amphibian system--a finding that has important implications in conservation of biodiversity within this threatened vertebrate taxon and whose mechanistics may have broader implications in biomolecular science.

Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the “shotgun” cloning of novel brevinins by means of 3'-RACE, using a “universal” degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.

Cloning from tissue surrogates: antimicrobial peptide (esculentin) cDNAs from the defensive skin secretions of Chinese ranid frogs

The defensive skin secretions of amphibians are a rich source of bioactive peptides. Here we describe a rapid technique for skin granular gland transcriptome cloning from a surrogate tissue-the secretion itself. cDNA libraries were constructed from lyophilized skin secretion from each of the Chinese frogs (Rana schmackeri, Rana versabilis, and Rana plancyi fukienensis) using magnetic oligo(dT) bead-captured polyadenylated mRNA as templates. Specific esculentin cDNAs were amplified by 3'-RACE using a degenerate primer designed for a consensus nucleotide sequence in the 5' untranslated region of previously characterized ranid frog peptide cDNAs. The cloned cDNAs were found to encode the antimicrobial peptides esculentins 1 and 2 from each of the species examined. The presence of predicted peptide structures in skin secretions was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This experimental approach can thus rapidly expedite parallel transcriptome and peptidome analysis of amphibian granular gland secretions without harming or sacrificing donor animals.