基于诊断引物的赤眼蜂鉴定和检测技术

通过对松毛虫赤眼蜂Tichogramma dendrolimi Matsumura和玉米螟赤眼蜂T.ostriniae Pang et Chen（膜翅目：赤眼蜂科）核内可转录第二间隔区（简称：ITS2）的克隆、测序,并获取和分析了GenBank中已登录的同源序列,然后设计了松毛虫赤蜂的特异引物以用于该蜂的分子鉴定和检测,经过反复筛选发现：采用鉴定引物通过PCR扩增不仅可以区分鑫头样品,单头样品（雌蜂或雄峰）,而且可鉴定幼期虫和卵,这用传统方法是无法办到的。该鉴定技术比基于形态学鉴定检测技术用来鉴定了从中国大陆不同地域和寄主上采集到的12个样品,结果表明：该方法可用于赤眼蜂田间分子监测和实验室拟寄生行为研究。     

诊断引物应用于我国三种重要赤眼蜂分子鉴定的研究

本研究根据螟黄赤眼蜂rDNA-ITS2序列设计了螟黄赤眼蜂Trichogramma chilonis Ishii特异引物,同时采用文献中发表的松毛虫赤眼蜂Trichogramma dendrolimi Matsumura 和玉米螟赤眼蜂Trichogramma ostriniae Pang et Chen的特异引物以及赤眼蜂属Trichogramma 特异引物对赤眼蜂成虫和寄主卵样品进行了PCR特异扩增分析。结果表明,采用上述特异引物可从单头蜂稳定地扩增出明显的目的DNA条带,并且分子鉴定结果与形态学鉴定结果完全吻合。因此,采用上述3对特异引物可以实现对我国3种重要赤眼蜂种,即松毛虫赤眼蜂、螟黄赤眼蜂和玉米螟赤眼蜂的分子鉴定。     

赤眼蜂分子鉴定技术研究

通过对6种常见赤眼蜂,即松毛虫赤眼蜂Trichogramma dendrolimi Matsumura、玉米螟赤眼蜂T. ostriniae Pang et Chen、螟黄赤眼蜂T. chilonis Ishii、广赤眼蜂T. evanescens Westwood、甘蓝夜蛾赤眼蜂T. brassicae Bezdenko及食胚赤眼蜂T. embryophagum (Hartig)之核糖体核糖核酸基因第二内部转录区（rDNA-ITS2）的克隆测序,调用GenBank中同源序列,对不同蜂种的rDNA-ITS2序列进行了多重排比和聚类,探讨了rDNA-ITS2用于赤眼蜂属不同种系统进化关系分析及赤眼蜂分子鉴定的可行性。为了考察rDNA-ITS2在赤眼蜂种下水平鉴定上的可能性,作者收集了我国常见的松毛虫赤眼蜂6个地理种群（黑龙江亚布力、吉林长春、吉林仁和、陕西长安、江苏徐州、广东广州）,采用相同方法测定了它们的rDNA-ITS2序列。序列分析结果表明,赤眼蜂种下阶元ITS2序列非常保守,而种间存在明显的遗传差异。通过外群比较发现,rDNA-ITS2只适合于赤眼蜂种一级的分子鉴定。     

不同赤眼蜂对灰白蚕蛾卵的偏好性比较

在实验室条件下,利用不同种类和品系的赤眼蜂Trichogramma spp.对灰白蚕蛾Ocinnara varians选择性和灰白蚕蛾对其适合性的差异,筛选防治灰白蚕蛾的有效蜂种和品系,结果表明,在供试的8种赤眼蜂中,松毛虫赤眼蜂Trichogramma dendrolimi、短管赤眼蜂Trichogramma pretiosum、舟蛾赤眼蜂Trichogramma closterae、暗黑赤眼蜂Trichogramma euproctidis和拟澳洲赤眼蜂Trichogramma confusum 5种赤眼蜂较其它3种赤眼蜂更偏好寄生灰白蚕蛾的卵,每雌蜂在灰白蚕蛾卵上的接触次数、寄生卵数都相对较多,且羽化率也都较高.广赤眼蜂Trichogramma evanescens对灰白蚕蛾卵的接触次数最多,为6.60次/雌,但寄生卵数却很少,为4.55粒/雌,羽化率也很低,为27.86%.玉米螟赤眼蜂寄生灰白蚕蛾的卵比较多,为14.35粒/雌,但对灰白蚕蛾卵的接触次数很少,仅1.20次/雌,羽化率也很低,为24.17%.卷蛾分索赤眼蜂对灰白蚕蛾卵的接触次数和寄生卵数最少的,分别为0.60次/雌和2.20粒/雌,且被寄生卵全部不能发育成功.综合分析结果表明,松毛虫赤眼蜂是最适合寄生灰白蚕蛾卵的赤眼蜂种,可作为其生物防治的潜在寄生蜂.     

短期高温对感染Wolbachia的松毛虫赤眼蜂发育和繁殖的影响

感染了沃尔巴克氏体Wolbachia的松毛虫赤眼蜂Trichogramma dendrolimi Matsumura营孤雌产雌生殖方式,但高温可能影响这一特性。为了模拟该赤眼蜂在田间遇到短期高温后的受影响情况,研究了柞蚕卵繁殖的Wolbachia感染了Wolbachia的松毛虫赤眼蜂孤雌产雌品系,于蛹中期和蛹后期经历6h32,35,38℃高温单次冲击处理,对当代羽化出蜂率、单卵出蜂数、单卵雌蜂率、寿命4个指标和处理子代羽化出蜂率、有效繁殖个体、单卵出蜂数、单卵雌蜂率4个指标的影响。结果表明,短期高温刺激会对孤雌产雌的松毛虫赤眼蜂产生不利影响,尤其是38℃高温不利影响明显,主要表现在羽化出蜂率和单卵出蜂数两指标明显降低;高温冲击对子代蜂各指标影响不明显。2代赤眼蜂均未有雄峰出现,表明短期高温冲击不能对Wolbachia调控其宿主生殖方式的作用产生影响。     

玉米螟赤眼蜂种群特异分子标记构建及其应用

赤眼蜂是我国乃至世界范围内应用最广的卵寄生蜂,其中玉米螟赤眼蜂Trichogramma ostriniae Pang et Chen是我国玉米种植区重要的天敌昆虫,但其不同的地理种群在生物防治效果上存在较大差异。为了有效利用玉米螟赤眼蜂的优势种群,有必要对其进行准确的鉴别。然而,采用传统形态学方法对同一种赤眼蜂的不同种群进行鉴别是非常困难的。本研究首先采用随机扩增多态性DNA(RAPD)标记对采自我国北京农科院、山东日照和山西太原的3个玉米螟赤眼蜂种群,进行了遗传差异分析,然后筛选了种群特异性位点,并通过克隆测序和引物设计构建了种群特异性分子标记,即序列特征性扩增区域(SCAR)标记。最后,利用SCAR标记对混合蜂群竞争试验结果进行了分子检测。该研究方法有助于玉米螟赤眼蜂优势种群的筛选,对于赤眼蜂的有效利用具有重要的意义。     

安荔赤眼蜂在中国野外首次发现及其体内Wolbachia的检测

【目的】为丰富赤眼蜂Trichogramma的种类资源,明确野外新采集的一种赤眼蜂的种类,探明该赤眼蜂所感染Wolbachia的类型。【方法】采用挂米蛾Corcyra cephalonica卵卡法在华南农业大学树木园诱集到两批赤眼蜂,通过形态鉴定和PCR扩增ITS2序列并测序分析的分子鉴定手段对野外采集的赤眼蜂材料进行种类鉴定;通过PCR扩增Wolbachia的外膜蛋白基因(wsp)序列,检测赤眼蜂体内Wolbachia的感染情况;通过PCR扩增wsp序列和多位点序列分型(multilocus sequence typing, MLST)对检测到的赤眼蜂体内的Wolbachia进行同源性分析。【结果】所诱集到的两批赤眼蜂均被鉴定为安荔赤眼蜂Trichogramma oleae Voegelé & Pointel,体内Wolbachia的感染率达100%。该Wolbachia株系与安荔赤眼蜂(前南斯拉夫品系)、短管赤眼蜂T. pretiosum(乌拉圭品系)以及T. deion(荷兰品系)体内Wolbachia亲缘关系较近,属于B超组Sib亚组,对应MLST序列型为ST486。【结论】安荔赤眼蜂T. oleae为中国野外首次发现,是完全感染Wolbachia的产雌孤雌生殖品系。本研究为害虫生物防治提供了一种新的天敌种类资源,并为进一步探明Wolbachia与赤眼蜂的互作提供了研究材料。     

基于分子生物学方法的外来入侵物种入侵历史重构

生物入侵是一个世界性的问题。全球每年因生物入侵造成的损失超过1万亿美元。探究入侵物种在入侵地的入侵历史对了解生物入侵的生物生态学机制、制定阻截及防除措施有重要意义。分子标记方法的兴起和大规模应用打开了入侵生物入侵历史研究的新天地。采用分子标记的方法可鉴定入侵物种的种类、追溯其来源地、回溯其扩散路径、分析扩散模式及探究物种入侵过程中对入侵种群本身的变化及其对生态系统所造成的各种影响。分子标记的应用使得多个入侵物种的入侵历史得以重现。由于分子标记方法重构的入侵历史受采样范围、采用的分子标记的种类及数量等因素的影响,该方法呈现入侵历史是否是真实发生的入侵过程还存在争议。     

寄生玉米螟卵赤眼蜂种类的鉴别

近几年北方玉米田广泛利用赤眼蜂防治玉米螟,地区间相互引进蜂种,其中存在着选用合宜的蜂种以获取较好的防治效果问题。1974年我们在开门办学中,深入农村,调查了天津自然界寄生玉米螟卵的赤眼蜂种类,同时鉴定了自河南省开封县、兰考县引入天津防治玉米螟的赤眼蜂,以及河北省博野县、唐山市郊区为防冶玉米螟繁殖的赤眼蜂。我们观察到天津自     

松毛虫赤眼蜂两性生殖品系和孤雌产雌品系在不同品种柞蚕卵中的寄生和生长发育表现

【目的】明确两性生殖品系和孤雌产雌品系松毛虫赤眼蜂Trichogramma dendrolimi在不同品种柞蚕Antheraea pernyi卵中的寄生及发育表现,为以柞蚕卵为替代寄主更好地规模化繁育赤眼蜂提供依据。【方法】测定两性生殖品系和孤雌产雌品系雌蜂在6个品种柞蚕卵［抗大(KD)、大四(DS)、高新(GX)、988(NEE)、青大(QD)和特大(TD)］上的寄生率、子代蜂窝蜂数(单窝羽化子代蜂数)和子代蜂雌性比等生物学指标,以及6个品种柞蚕卵的质量指标(单卵湿重、蛋白质含量、总糖含量和甘油三酯含量);利用主成分分析揭示柞蚕卵质量指标与赤眼蜂生物学指标间的相关性。【结果】NEE品种柞蚕卵育出的两性品系松毛虫赤眼蜂子代蜂个体显著大于除KD和QD品种以外的其他柞蚕品种卵育出的两性品系子代蜂。柞蚕品种对两性品系松毛虫赤眼蜂的寄生率、子代蜂窝蜂数和子代蜂雌性比均无显著影响。在孤雌产雌品系松毛虫赤眼蜂中,KD品种柞蚕卵育出的松毛虫赤眼蜂子代蜂窝蜂数最多,显著高于NEE和QD品种育出的子代蜂窝蜂数,但与DS, GX和TD品种育出的子代蜂窝蜂数相比无显著差异。柞蚕品种对孤雌产雌品系松毛虫赤眼蜂的寄生率和子代蜂个体大小均无显著影响。TD品种柞蚕卵的蛋白质含量最高,显著高于除GX品种以外的柞蚕品种。KD品种柞蚕卵的总糖含量和QD品种柞蚕卵的甘油三酯含量最高,均分别显著高于其余柞蚕品种。主成分分析表明,两性品系松毛虫赤眼蜂寄生率、柞蚕卵蛋白质含量和甘油三酯含量均与柞蚕卵总糖含量呈负相关;两性品系松毛虫赤眼蜂子代蜂个体大小和柞蚕卵甘油三酯含量与单卵湿重呈负相关;孤雌产雌品系松毛虫赤眼蜂寄生率和柞蚕卵甘油三酯含量与松毛虫赤眼蜂子代蜂窝蜂数、柞蚕卵总糖含量和单卵湿重呈负相关;孤雌产雌品系松毛虫赤眼蜂子代蜂个体大小和柞蚕卵总糖含量与蛋白质含量呈负相关。【结论】本研究通过筛选适宜两性生殖品系和孤雌产雌品系赤眼蜂繁育的柞蚕卵品种,初步明确了卵内主要营养指标与子代蜂生物学指标间的相关性。研究结果为利用柞蚕卵更好地规模化繁育松毛虫赤眼蜂提供依据与数据支撑。     

M-PCR： a powerful method for rapid molecular identification of Trichogramma wasps （Hymenoptera： Trichogrammatidae）

Two species-specific primers were designed depending on ITS2 sequence variation of 37 Trichogramma wasps, and these primers were applied to establish an assay,multiplex PCR (M-PCR), for molecular diagnosis of two important Trichogramma wasps,T. confusum and T. dendrolimi, in China. Multiplex-PCR results showed that only target species produced two PCR products, one product of ITS2 region species-specific amplification and one product of its ITS 1 region universal amplification, but other species produced only one ITS1 universal PCR product. Using this method, the target Trichogramma species can be distinguished from other Trichogramma species. Molecular identification based on M-PCR has particular value over morphological technology and other approaches, such as normal molecular and biochemical methods. Furthermore, because M-PCR assay can avoid false negative results, which frequently happen in PCR reaction, this method will be much more accurate and useful for Trichogramma identification, and can be developed as an easy and rapid diagnostic kit applied in the identification and quality monitoring of Trichogramma mass products both in the factory and in the field. Such an easy and rapid diagnostic kit will be valuable in the application of Trichogramma species as a biological control.     

A rapid single-step multiplex method for discriminating between Trichogramma (Hymenoptera: Trichogrammatidae) species in Australia

Inaccurate species identification confounds insect ecological studies. Examining aspects of Trichogramma ecology pertinent to the novel insect resistance management strategy for future transgenic cotton, Gossypium hirsutum L., production in the Ord River Irrigation Area (ORIA) of Western Australia required accurate differentiation between morphologically similar Trichogramma species. Established molecular diagnostic methods for Trichogramma identification use species-specific sequence difference in the internal transcribed spacer (ITS)-2 chromosomal region; yet, difficulties arise discerning polymerase chain reaction (PCR) fragments of similar base pair length by gel electrophoresis. This necessitates the restriction enzyme digestion of PCR-amplified ITS-2 fragments to readily differentiate Trichogramma australicum Girault and Trichogramma pretiosum Riley. To overcome the time and expense associated with a two-step diagnostic procedure, we developed a "one-step" multiplex PCR technique using species-specific primers designed to the ITS-2 region. This approach allowed for a high-throughput analysis of samples as part of ongoing ecological studies examining Trichogramma biological control potential in the ORIA where these two species occur in sympatry.     

Molecular genetics and the management and conservation of marine organisms

Biochemical and molecular species identification techniques have a broad range of applications in the management and conservation of marine organisms. While species boundaries are not always clearly defined, phylogeneticists utilise autapomorphic characters to distinguish phylogenetic species. Genetic markers discriminate between marine taxa when traditional morphological distinctions are unclear. The applications of these techniques can be divided into four general categories. Firstly, compliance enforcement, which often depends on genetic identification techniques to enable officials to identify the species to which regulations pertain. Secondly, quality control applications, to allow for the testing of marine products to guard against fraudulent substitution with less valuable species, which is particularly pertinent since processing often obliterates identifiable features. Thirdly, a variety of applications to ecological and life-history studies and conservation management are reported. Here, the genetic identification techniques of species from cryptic life-cycle stages or of morphologically indistinct species are an indispensable tool for marine scientists, conservators and managers. Lastly, the application of genetic techniques for sourcing population origin is briefly discussed. The biochemical and molecular techniques applied to species identification all exploit phenotypic or genotypic polymorphisms that are sampled using either tertiary level protein based methods or primary level DNA based methods. In this review, examples of the applications along with the total protein, allozyme, serological, PCR and other DNA based methodologies are briefly described and some generalities with regard to their use are presented.     

A novel approach for the identification of bacterial taxa-specific molecular markers

AIMS: To develop and establish a methodology for an oriented and fast identification of species taxa-specific molecular markers useful for the identification of micro-organisms. METHODS AND RESULTS: From the complete microbial genomes available in Pfam database, taxa-specific protein domains were identified which lead to the selection of taxa-specific loci. This strategy was used to identify six genetic markers: four specific for Pseudomonas syringae pv. tomato, one specific for P. syringae pv. syringae and one specific for P. putida. The discriminatory potential of these loci was evaluated by Southern hybridization using several pseudomonad species and pathovars, by dot-blot hybridization and by multiplex PCR optimized for the simultaneous detection of P. putida, P. syringae pv. syringae and P. syringae pv. tomato. Sensitivity assays indicated a detection limit of approximately 10 pg of chromosomal DNA template needed for each bacterium. CONCLUSIONS: The proposed methodology was efficient on the selection of six Pseudomonas-specific markers able to discriminate Pseudomonas at the species and pathovar level. SIGNIFICANCE AND IMPACT OF THE STUDY: The oriented search of taxa-specific molecular probes described in this work, which can be easily extended to other groups of bacteria, will improve the accuracy and expedite the identification of micro-organisms by DNA-based molecular methods.     

PCR-BASED TECHNIQUE FOR IDENTIFICATION AND DETECTION OF TRICHOGRAMMA SPP. (HYMENOPTERA: TRICHOGRAMMATIDAE) WITH SPECIFIC PRIMERS

Abstract The rDNA-ITS2 regions of T. dendrolimi Matsumura and T. ostriniae Pang et Chen (Hymenoptera: Trichogrammatidae) were cloned and sequenced. The homologous sequences available in GenBank were retrieved and analyzed, and then specific primers were designed for molecular identification and detection of T. dendrolimi . Repeated screening showed that PCR amplification by the diagnostic primers enabled the differentiation of not only bulk samples and single adult (male or female), but also eggs and juveniles, which was not possible by conventional methods. The advantage of this system over morphology-based systems is that non-specialists are able to identify individuals or trace specimens efficiently. The derived molecular detection technique was then used to identify 12 specimens collected from different localities on the Chinese mainland; the results showed that this protocol could be applied to molecular monitoring of Trichogramma species in the field. Finally, 1132s of 6 geographical populations of T. dendrolimi (TdCHA, TDJL, TdXZ, TdKH, TdCZ and TdYBL) were cloned and sequenced. The multialignment analysis of intraspecific ITS2 sequences showed that the diagnostic primers have their own theoretical bases.     

昆虫病原线虫资源概况和分类技术进展

昆虫病原线虫是具有重要潜在应用价值的害虫生物防治资源,主要包括斯氏线虫科（Steinernematidae）的斯氏线虫属Steinernema与新斯氏线虫属Neosteinernema线虫和异小杆线虫科（Heterorhabditidae）的异小杆线虫属Heterorhabditis线虫。近10年来,分子生物学方法与传统的形态学方法相结合应用到线虫的鉴定与分类,昆虫病原线虫的分类进入稳定与发展时期,越来越多的新种或品系被发现及应用于生物防治。目前已描述的昆虫病原线虫种类达65种,其中斯氏线虫属52种,新斯氏线虫属1种,异小杆线虫属12种。本文整理列出了迄今报道的昆虫病原线虫种类及其来源,并综述了昆虫病原线虫分类现状以及鉴定与分类方法上的研究进展,重点阐述了分子生物学技术在昆虫病原线虫鉴定与分类的应用状况。     

Multilocus species identification and fungal DNA barcoding: insights from blue stain fungal symbionts of the mountain pine beetle

There is strong community-wide interest in applying molecular techniques to fungal species delimitation and identification, but selection of a standardized region or regions of the genome has not been finalized. A single marker, the ribosomal DNA internal transcribed spacer region, has frequently been suggested as the standard for fungi. We used a group of closely related blue stain fungi associated with the mountain pine beetle (Dendroctonus ponderosae Hopkins) to examine the success of such single-locus species identification, comparing the internal transcribed spacer with four other nuclear markers. We demonstrate that single loci varied in their utility for identifying the six fungal species examined, while use of multiple loci was consistently successful. In a literature survey of 21 similar studies, individual loci were also highly variable in their ability to provide consistent species identifications and were less successful than multilocus diagnostics. Accurate species identification is the essence of any molecular diagnostic system, and this consideration should be central to locus selection. Moreover, our study and the literature survey demonstrate the value of using closely related species as the proving ground for developing a molecular identification system. We advocate use of a multilocus barcode approach that is similar to the practice employed by the plant barcode community, rather than reliance on a single locus.