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.     

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.     

A specific PCR assay for the diagnosis of Clonorchis sinensis infection in humans, cats and fishes

Clonorchiasis caused by Clonorchis sinensis is a fish-borne parasitic disease which is endemic in a number of countries. Using the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of C. sinensis as genetic markers, a pair of C. sinensis-specific primers was designed and used to establish a specific PCR assay for the diagnosis of C. sinensis infection in humans, cats and fish. This approach allowed the specific identification of C. sinensis after optimizing amplification conditions, with no amplicons being amplified from related heterogeneous DNA samples, and sequencing of amplicons confirmed the identity of the sequences amplified. The detection limit of this assay was 1.03 pg of adult C. sinensis, 1.1 metacercariae per gram of fish filet, and a single egg in human and cat feces. The PCR assay should provide a useful tool for the diagnosis and molecular epidemiological investigation of clonorchiasis in humans and animals.     

中华鲟抗菌肽Hepcidin的克隆、表达及其抗菌活性分析

Hepcidin也称为铁调素,是在肝脏中特异表达的一种阳离子小分子抗菌肽。2000年,Krause,et al.首先从人血液中分离纯化得到了由25个氨基酸组成的LEAP-1抗菌肽[1]。2002年,Shike,et al.首次从杂交条纹鲈的鳃分离出鱼类Hepcidin,并从金眼狼鲈(Morone chrysops)克隆到     

不同盐度条件下中华绒螯蟹亲蟹行为及血淋巴生理变化

设定淡水对照组、盐度18适应组、盐度30骤变组(18→30)和盐度0骤变组(18→0),采用视频记录分析法研究了不同盐度条件下中华绒螯蟹雌性亲蟹的8项行为学指标变化,并测定了血淋巴渗透压及离子、血蓝蛋白含量。结果表明:中华绒螯蟹亲蟹封闭反应行为仅发生于盐度组(盐度18组和盐度30骤变组),且盐度30骤变组封闭反应时间显著高于盐度18组(P<0.05);腹部开合行为仅见于盐度0骤变组;盐度组第一触角回缩时间显著高于对照组(P<0.05);其他5项行为学指标活动频率均于盐度0骤变组最高。各实验组亲蟹血淋巴渗透压及离子浓度均高于外界实验水体,且均随盐度升高而增大。血蓝蛋白含量随盐度的降低而升高,盐度0骤变组血蓝蛋白含量显著高于盐度30骤变组(P<0.05)。分析认为,中华绒螯蟹亲蟹在0～30盐度范围内进行高渗透压调节,腹部开合行为是其在低盐度环境下暴露尾肠吸收水中离子的一种行为策略,封闭反应有助于机体减少高盐度坏境下水的吸收及扩散失盐。     

至少6个突变基因型冬虫夏草菌在冬虫夏草子座中表达的动态变化

本研究检测了在冬虫夏草成熟过程中突变基因型冬虫夏草菌在子座中表达的动态变化。根据冬虫夏草菌基因内转录间隔区(ITS)序列中大量散在的点突变,设计了8个单核苷酸多态性(SNP)延伸引物,采用SNP质谱基因分型法测定各个SNP位点的单核苷酸延伸反应,观察冬虫夏草子座中转换突变和颠换突变基因型菌在冬虫夏草成熟过程中表达的动态变化。其中5个SNP延伸引物(067721-211,067721-240,067721-477,067721-531和067721-581)位于rDNA ITS1和ITS2段,用于区分GC和AT偏倚基因型,但不区分2个AT偏倚基因型。另外3个延伸引物(067740-324,067740-328和067740-360)位于rDNA 5.8S段,用于区分2个AT基因型。采用PCR+EcoRⅠ限制性酶切法检验2个GC偏倚基因型冬虫夏草菌在冬虫夏草成熟过程中表达的动态变化。结果表明:冬虫夏草子座rDNA ITS1-5.8S-ITS2片段的8个SNP位点的质谱图谱显示冬虫夏草子座中存在至少5个冬虫夏草菌转换突变和颠换突变等位基因。它们的表达在冬虫夏草成熟过程中呈现动态变化。067721-211和067721-477位点的AT偏倚型等位基因的峰高明显高于GC偏倚型;随着冬虫夏草的成熟,这2个位点的AT型等位基因峰高大幅度下降。SNP质谱图不仅显示转换突变的等位基因,颠换突变等位基因也有很高的检出率,它们的峰高在一些位点甚至超过GC和AT基因型等位基因;随着冬虫夏草的成熟,颠换突变等位基因的检出率和峰高趋于下降。在区分2个AT偏倚基因型的研究中,AB067744和AB067740 2个基因型的等位基因同时存在于未成熟冬虫夏草子座中。随着冬虫夏草的成熟,AB067744基因型等位基因的峰高明显降低,而AB067740基因型等位基因的峰高明显升高。PCR产物EcoRⅠ酶切试验发现子座中存在2组GC偏倚型菌,具有完全不同的成熟模式。综上,冬虫夏草子座中存在至少6个突变基因型冬虫夏草菌,其表达随着冬虫夏草的成熟呈现动态变化。这些突变基因型菌表达的动态变化对于冬虫夏草子座的萌发和成熟具有重要菌物学意义。     

茶树α-tubulin基因实时荧光定量RT-PCR方法的建立

目的:建立茶树α-tubulin基因实时荧光定量RT-PCR方法。方法:根据GenBank中茶树α-tubulin基因保守区域设计一对特异性引物,将PCR扩增得到的α-tubulin基因克隆到pTG19-T载体上,构建的重组质粒标准品经1/10梯度稀释后,用SYBR GreenⅠ染料法绘制标准曲线,并进行融解曲线分析。结果:标准曲线Ct值检测范围为14.56~27.09,相关系数为0.991,溶解曲线分析显示产物为特异的单峰,其Tm值为81±0.3℃。结论:建立了茶树α-tu bulin基因实时荧光定量RT-PCR法,为以α-tubulin作为茶树内参基因进行功能基因表达差异研究奠定了基础。     

中华水韭残存居群的数量性状分化和地方适应性及其对保育遗传复壮策略的提示

 研究残存居群的数量性状分化及其地方适应性有助于了解不同居群对环境的适应性并制定相应的保育策略。本研究采用同园实验,利用随机区 组设计,对中华水韭(Isoetes s inensis)3个居群的9种优势基因型的数量性状进行巢式方差分析,并采用Bayesian方法计算等位酶（F_ST）和 数量性状（Q_ST）的居群分化值。结果表明,在测量的14个数量性状中,有10个性状在居群间有显著性差异,仅有3个性状在居群内有显著性差 异;多重比较表明其中5个性状的平均值均以松阳居群最高,休宁居群最低;建德居群的大孢子囊的性状和植株高度的平均值最高,休宁居群最 低;推测这可能是与奠基者效应、物种间竞争及中华水韭在自然生境下形成的营养生长和生殖生长之间的权衡有关。对居群数量性状分化的QST 值和等位酶分化的FST值分析比较发现,在假设为自交系时有8个性状的Q_ST值均大于F_ST值,表明存在明显的地方适应性,居群间个体的混合存 在潜在的远交衰退的风险,所以不宜采用居群间相互移植个体的方法来进行中华水韭居群的遗传复壮。对休宁和松阳两个居群的保育应采用提 高居群内基因流和改善生境环境的方法,促进居群内的遗传复壮;对建德居群应尽量减少人为干扰,宜建立特定生境的保护小区。     

广东海丰鸟类自然保护区褐翅鸦鹃数量调查

为掌握褐翅鸦鹃(Centropus sinensis)在广东海丰鸟类自然保护区的种群现状,提出有针对性的保护措施,2005年对保护区内褐翅鸦鹃数量行了调查.4个季节中,夏季记录的个体数最多(32只),其样线平均个体数与春季、秋季相比,差异性显著(P＜0.05);夏季种群密度最大,达17.78只,km2,冬季仅为1.11只/km2.单次记录到的多为单只个体.不同季节在3个保护站记录到的褐翅鸦鹃数量存在一定的差异.总体上,东关联安围记录到的个体数最多.