Genetic consequence of restricted habitat and population decline in endangered Isoetes sinensis (Isoetaceae)

BACKGROUND AND AIMS: Isoetes sinensis (Isoeteaceae) is a critically endangered aquatic quillwort in eastern China. Rapid decline of extant population size and local population extinction have occurred in recent years and have raised great concerns among conservationists. METHODS: Amplified fragment length polymorphisms (AFLPs) were used to investigate the genetic variation and population structure of seven extant populations of the species. KEY RESULTS: Eight primer combinations produced a total of 343 unambiguous bands of which 210 (61.2 %) were polymorphic. Isoetes sinensis exhibited a high level of intra-population genetic diversity (H(E) = 0.118; hs = 0.147; I = 0.192; P = 35.2 %). The genetic variation within each of the populations was not positively correlated with their size, suggesting recent population decline, which is well in accordance with field data of demographic surveys. Moreover, a high degree of genetic differentiation (F(ST) = 0.535; G(ST) = 0.608; theta(B) = 0.607) was detected among populations and no correlation was found between geographical and genetic distance, suggesting that populations were in disequilibrium of migration-drift. Genetic drift played a more important role than gene flow in the current population genetic structure of I. sinensis because migration of I. sinensis is predominantly water-mediated and habitat range was highly influenced by environment changes. CONCLUSIONS: Genetic information obtained in the present study provides useful baseline data for formulating conservation strategies. Conservation management, including both reinforcement for in situ populations and ex situ conservation programmes should be carefully designed to avoid the potential risk of outbreeding depression by admixture of individuals from different regions. However, translocation within the same regional population should be considered as a measure of genetic enhancement to rehabilitate local populations. An ex situ conservation strategy for conserving all extant populations to maximize genomic representation of the species is also recommended.     

Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants （Camellia sinensis （L.） O. Kuntze）

β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity,with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50℃ and was stable at temperatures lower than 40℃. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.     

茶树鲜花饮料澄清技术研究

茶树鲜花与茶叶具有相似的营养成分,榨汁后经调配可开发成适口性好、风味独特的鲜花饮料。对茶树鲜花饮料的壳聚糖絮凝、酶法澄清、超滤澄清效果进行了比较,结果表明,采用10万分子截留量的超滤膜处理获得能够较好的澄清效果,且对茶多酚造成的操作损失较小。     

蝎蛉科与蚊蝎蛉科昆虫触角感器超微形态比较

触角是昆虫重要的感觉器官,其感觉功能主要是通过其上的各类感器来实现的。本文以染翅蝎蛉Sinopanorpa tincta(Nav偄s)和中华蚊蝎蛉Bittacus sinensis Walker为代表,采用扫描电子显微镜观察了蝎蛉科Panorpidae和蚊蝎蛉科Bittacidae触角感器的类型及其超微结构,并简要讨论了各种感器的功能。结果显示:染翅蝎蛉触角上共发现7种感器,分别为短刺形感器、Bhm氏鬃毛、毛形感器、短毛形感器、锥形感器、刺形感器、和微毛形感器。中华蚊蝎蛉触角上也发现7种感器,分别为Bhm氏鬃毛、芽形感器、毛形感器、刺形感器、畸形毛感器、腔锥感器和钟形感器。蚊蝎蛉触角与蝎蛉的最大区别是着生有大量的腔锥感器,初步推测众多的腔锥感器可能与蚊蝎蛉对湿度的高度敏感有关。     

雄全异株植物瘿椒树（省沽油科）的传粉生物学

从开花动态、传粉昆虫、花的形态结构、繁育系统、花粉活力和柱头可授性等方面研究了我国特有珍稀植物瘿椒树（Tapiscia sinensis Oliv.）的传粉生物学特性。瘿椒树是典型的雄全异株植物,两性花中含有功能性花粉,且自交亲和,但雄花花粉活力和萌发力是两性花的10倍以上。雄株和两性植株具有相同开花物候期,花期均为5月下旬至6月上旬,单花期为4-5天,雄花和两性花的5枚花药开裂的不同步性明显延长了散粉时间。两性花雌蕊先熟,柱头可授性较长。具有适应风媒和虫媒传粉的花部特征。传粉昆虫主要为蜜蜂科（Apidae）和食蚜蝇科（Syrphidae）昆虫,访花高峰期为8：30-10：30。维持瘿椒树雄全异株的可能机制是：雄株总体上增加了异交花粉的数量和质量;两性花的雄蕊为该物种提供了繁殖保障,同时为传粉者提供了报酬。     

不同产地中华鳖的线粒体控制区序列分析及结构比较

采用PCR特异引物,扩增了两产地中华鳖（Pelodiscus sinensis）个体的mtDNA控制区（CR）及其邻近片段,测序获得了长度分别为1830bp和1630bp的序列。结合GenBank中已发表的韩国产中华鳖mtDNA的CR区序列,比较了3个产地中华鳖的CR区结构。分析显示：中华鳖不同产地mtDNA CR区DNA中的A＋T含量分别为60.5%、63.6%和64.8%,它们的5′、3′末端以及CSB1-CSB2之间均存在丰富的可变数目串联重复序列（variable numbers oftandem repeats,VNTR）。基于mtDNA CR区序列和结构分析,显示中华鳖不同产地的野生个体中存在丰富的遗传多样性。     

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.     

A new loach, Cobitis shikokuensis (Teleostei: Cobitidae), from Shikoku Island, Japan

A new species of spinous loach, Cobitis shikokuensis, is described based on 297 specimens from Shikoku Island, Japan. The new species was formerly known as the Shikoku group of Cobitis takatsuensis. It can be distinguished from other species of Cobitis and closely related genera by a combination of the following characters: dorsal fin with 6 branched soft rays; anal fin with 5 branched soft rays; one brownish streak across eye from the tip of nose, no streak on cheek; a black spot smaller than eye diameter near the dorsal corner of the caudal fin base; 3–5 small brownish speckles on ventral side of caudal peduncle; high caudal peduncle with well-developed fleshy keels on dorsal and ventral side; a lamina circularis at base of dorsal part of pectoral fin absent; first branched soft ray of pectoral fin broad in males; pectoral soft rays widely branched from the approximate midpoint; last anal fin ray with 2 elements; interorbital width 11.2–17.1% of head length.     

Mycelium cultivation, chemical composition and antitumour activity of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

AIMS: To examine and illustrate the morphological characteristics and growth kinetics of Cs-HK1, a Tolypocladium fungus, isolated from wild Cordyceps sinensis in solid and liquid cultures, and the major chemical constituents and antitumour effects of Cs-HK1 mycelium. METHODS AND RESULTS: The Cs-HK1 fungus was isolated from the fruiting body of a wild C. sinensis and identified as a Tolypocladium sp. fungus. It grew rapidly at 22-25 degrees C on a liquid medium containing glucose, yeast extract, peptone and major inorganic salts, with a specific growth rate of 1.1 day(-1), reaching a cell density of 23.0 g dw l(-1) in 7-9 days. Exopolysaccharides accumulated in the liquid culture to about 0.3 g l(-1) glucose equivalent. In comparison with natural C. sinensis, the fungal mycelium had similar contents of protein (11.7-microg) and carbohydrate (654.6-microg) but much higher contents of polysaccharide (244.2 mg vs 129.5 mg), adenosine (1116.8-microg vs 264.6 microg) and cordycepin (65.7 microg vs 20.8 microg) (per gram dry weight). Cyclosporin A, an antibiotic commonly produced by Tolypocladium sp., was also detected from the mycelium extract. The hot water extract of mycelium showed low cytotoxic effect on B16 melanoma cells in culture (about 25% inhibition) but significant antitumour effect in animal tests, causing 50% inhibition of B16 cell-induced tumour growth in mice. CONCLUSIONS: The Tolypocladium sp. fungus, Cs-HK1, can be easily cultivated by liquid fermentation. The mycelium biomass contained the major bioactive compounds of C. sinensis, and the mycelium extract had significant antitumour activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The Cs-HK1 fungus may be a new and promising medicinal fungus and an effective and economical substitute of the wild C. sinensis for health care.     

Gene mapping of 28S and 5S rDNA sites in the spined loach Cobitis taenia (Pisces, Cobitidae) from a diploid population and a diploid–tetraploid population

We compare the chromosomal 28S and 5S rDNA patterns of the spined loach C. taenia (2n = 48) from an exclusively diploid population and from a diploid–polyploid population using 28S and 5S rDNA probe preparation and labelling, and fluorescence in situ hybridization (FISH). The 5S rDNA was located in two to three chromosome pairs, and separated from the 28S loci for the males and one female (F1) from the diploid population. Loaches from a diploid–polyploid population, and one female (F2) from the diploid population were characterized by at least one chromosome pair with 5S and 28S overlapping signals. The fishes differed mainly in their number of 28S rDNA loci, located on 3–6 chromosomes. All individuals from both populations were characterized by one acrocentric chromosome bearing a 28S rDNA signal on the telomeres of its long arm. The number of major ribosomal DNA in the karyotype of C. taenia by FISH was always higher than the number of Ag-NORs. Our data confirm the extensive polymorphism of NORs in both populations, as already has been observed in closely related Cobitis species, and less polymorphic 5S rDNA pattern. However, this preliminary result highlights the need for a wider scale study.