有效抽提乙醇中螨标本的核DNA

对如何有效地从乙醇保存的叶螨中抽提核基因组DNA作了探讨。70％乙醇保存的叶螨标本,经过干燥,TEN洗涤,蛋白酶K消化,酚—氯仿抽提,2倍体积无水乙醇沉淀等步骤,得到叶螨核基因组DNA沉淀,再用TE或无菌水溶解后,以其为模板,进行随机引物PCR扩增。结果显示.该方法抽提的DNA无Taq酶抑制物,且其纯度达到进行PCR反应对模板要求的程度。这为采用AP-PCR技术研究螨的系统分类提供了便利。     

螟卵啮小蜂产卵行为利它素的研究:存在部位和抽提

本文是有关螟卵啮小蜂产卵行为利它素研究的第一部份,报道了该利它素在寄主三化螟、白螟中的存在部位和抽提方法。 试验表明:刺激螟卵啮小蜂搜索寄主和产卵行为的利它素存在于寄主卵块的卵毛和雌蛾的尾毛之中,它能被丙酮—水(1:1)溶剂所抽提,但不能被正己烷所抽提。当把白螟卵毛的丙酮—水抽提浓缩物滴加到纸质模拟卵上后,能引起雌蜂对模拟卵的触角敲打和产卵管插入。     

甲氰菊酯与阿维菌素单用、轮用和混用对朱砂叶螨抗性进化的影响

应用数量遗传学的方法分析朱砂叶螨(Tetranychus cinnabarinus)实验种群对甲氰菊酯、阿维菌素及其混剂甲氰—阿维(甲氰菊酯:阿维菌素=8.9:0.1,m/m)的抗性现实遗传力,并测定了甲氰菊酯、阿维菌素分别连续单用、轮换使用、混合使用对朱砂叶螨抗性进化的影响。结果表明,筛选16代后,朱砂叶螨对甲氰菊酯、阿维菌素和甲氰—阿维的抗性现实遗传力分别为0.2853、0.1695和0.0804,朱砂叶螨对混剂的抗性现实遗传力低于对2个单剂的遗传力的一半,混用延缓抗性的效果将好于轮用。药剂连续单用、轮换使用和混合使用16代,朱砂叶螨对甲氰菊酯的抗性分别为28.52、28.03和10.81倍,对阿维菌素的抗性分别为3.24、2.82和1.41倍。朱砂叶螨对2种杀螨剂抗性进化速率为单用>轮用>混用,抗性测定结果表明甲氰菊酯与阿维菌素混用能有效延缓朱砂叶螨对2种药剂抗性的发展速率。     

缺铁逆境胁迫下水稻叶片蛋白的双向电泳及其蛋白质组图谱分析

水稻幼苗经缺铁胁迫诱导分别处理1、3、5天后,用酚法和TCA/丙酮法提取叶片中的可溶性蛋白进行双向电泳分析,从而研究在缺铁条件下叶片中蛋白表达的动态变化规律.结果显示1.不同pH IPG胶条分离蛋白的效果不同.用pH3-10的IPG胶条进行双向电泳,经考马斯亮蓝染色后,可在胶面上检测到大约450个蛋白点,其中约有89%的蛋白是酸性蛋白.如果用pH4-7的IPG胶条进行双向电泳,则可检测到大约600个蛋白点,其中有29个蛋白是上调表达,1个蛋白是下调表达,5个蛋白是诱导特异表达.2.不同方法提取的可溶性蛋白质量不同.TCA法简单易操作,似乎对于碱性蛋白的抽提效果更好,在2-DE图像上,减性端显示的蛋白点多;但此方法所得蛋白的再溶性差.酚法提取的蛋白再溶性好,所抽提的蛋白量较大,纯度较高.     

一种经济、高效的提取微生物胞内色素的方法

目的:探索建立一种经济、高效的直接抽提微生物胞内色素的方法.方法:收集刚进入生长平稳期的产生色素的菌株,菌体沉淀用灭菌PBS洗尽非生物体颗粒.菌体沉淀充分分散在丙酮里,随后把菌体在冰浴条件下行超声波裂解细胞,超声裂解功率与时间随菌体种类而变化,用丙酮萃取法获得色素粗产品.结果:采用超声波裂解萃取法获得了较好的抽提效果.结论:超声波裂解法是一种高效、经济简单的提取微生物胞内色素的好方法,值得推广.     

基于AFLP分析用吴茱萸叶高质量DNA的提取

目的：研究吴茱萸叶基因组DNA的提取方法,以用于扩增片段长度多态性（AFLP）分析。方法：设计了一种改良CTAB法：以石英砂代替液氮研磨;抽提前用不溶性PVP结合酚形成络合物,然后用缓冲液除去;抽提中加入Vc。将改良CTAB法所提吴茱萸叶DNA与传统的SDS法、CTAB法所提DNA进行比较。利用植物的核糖体DNA（rDNA）保守序列设计引物行PCR扩增鉴定吴茱萸DNA及其质量。并确定提取方法中最佳样本含量和β-巯基乙醇浓度。结果：改良CTAB法提取石虎、疏毛吴茱萸总DNA呈白色,A260/A280为1.721~1.886,DNA分子完整,约20kb左右,PCR扩增条带清晰、明亮,无杂带和脱尾。并确定0.10g为最佳样本量,2.0%为最佳β-ME浓度。结论：石英砂研磨简便、迅速、均匀,该实验所建立的改良CTAB法可有效避免次生代谢物的氧化褐变,是一种小量、快速提取吴茱萸叶DNA优化方法。     

一种简易快速高效提取麻疯树营养器官中RNA的方法

以麻疯树根、茎、叶为材料,用4种方法提取麻疯树中总RNA.琼脂糖凝胶电泳和紫外光谱分析结果表明,改良的张年辉法提取的RNA比张年辉法提取的完整性更好,时间更短,条件更粗放;试剂盒A提取的RNA多数降解;试剂盒B几乎不能提取其RNA.RT-PCR分析表明,以改良的张年辉法提取的根、茎、叶中RNA作模板也可成功地进行RT-PCR扩增.据此,我们认为用简易、快速、高效的改良张年辉法提取麻疯树根、茎、叶中RNA为最佳选择.     

一种新型叶螨饲养器的设计与制作

分析了现有的叶螨个体饲养或定量饲养的方法或器具的缺陷。根据酢浆草如叶螨Tetranychina harti(Ewing)及其寄主植物红花酢浆草(Oxalis corymbosa DC)的特点,设计、制作了一种新型饲养器,解决了常用的离体湿基叶片法中叶片表面温湿度与设定的环境条件不一致、叶螨生活状态不自然和活体叶片法中镜检观察不方便的问题,取得了较好的效果。本设计在其他叶螨或微小昆虫的饲养中也可以作为借鉴。     

截形叶螨药剂防治对比试验

截形叶螨(Tetranychus truncatus Ehara)在云南昆明主要为害大棚花卉,尤其对月季(Rosa chinensis)为害严重。使用玻片浸液法,选用6种化学杀螨剂和1种植物叶面保护剂对该叶螨进行室内药效测定;同时,选取真除螨、主力和植物叶面保护剂等3种杀螨剂实施田间防治试验。结果表明,室内施药48h后,真除螨2 000、3 000倍液杀螨效果达100%;田间喷药3d后,真除螨4 000倍液能达到30%的防治效果,可有效降低害螨数量。建议每年截形叶螨为害严重的5～11月期间,在保护地栽培的月季上连续施用真除螨。害螨发生高峰期前,可使用植物叶面保护剂,减少害螨的取食。     

螺螨酯对山楂叶螨的生物活性和防治效果

螺螨酯是一种高效、低毒、广谱性杀螨剂,为研究螺螨酯对山楂叶螨Tetranychus vinnensis各螨态的影响,用24%的螺螨酯悬浮剂分别处理山楂叶螨卵、若螨和成螨。试验结果表明,螺螨酯对螨卵和若螨活性高,其致死中浓度LC50分别为0.76mg/L和19.07mg/L。采用玻片浸渍法分别用螺螨酯48、96、192mg/L处理成螨,48h校正死亡率为2.6%、19.4%、19.4%。采用喷洒叶片法,分别用螺螨酯1.5、3、6、12、24和48mg/L处理成螨,成螨的繁殖力显著下降。在温室盆栽苹果上,药后21d螺螨酯60mg/L处理山楂叶螨的减退率为100%,显著高于对照药剂四螨嗪250mg/L的减退率33.3%。总之,螺螨酯对山楂叶螨各阶段都有良好的控制效果,可以作为防治该螨的推荐药剂,施药适宜时期为第1代卵期。     

A factorial design experiment as a pilot study for noninvasive genetic sampling

Noninvasive genetic sampling has increasingly been used in ecological and conservation studies during the last decade. A major part of the noninvasive genetic literature is dedicated to the search for optimal protocols, by comparing different methods of collection, preservation and extraction of DNA from noninvasive materials. However, the lack of quantitative comparisons among these studies and the possibility that different methods are optimal for different systems make it difficult to decide which protocol to use. Moreover, most studies that have compared different methods focused on a single factor – collection, preservation or extraction – while there could be interactions between these factors. We designed a factorial experiment, as a pilot study, aimed at exploring the effect of several collection, preservation and extraction methods, and the interactions between them, on the quality and amplification success of DNA obtained from Asiatic wild ass (Equus hemionus) faeces in Israel. The amplification success rates of one mitochondrial DNA and four microsatellite markers differed substantially as a function of collection, preservation and extraction methods and their interactions. The most efficient combination for our system integrated the use of swabs as a collection method with preservation at ?20 °C and with the Qiagen DNA Stool Kit with modifications as the DNA extraction method. The significant interaction found between the collection, preservation methods and the extraction methods reinforces the importance of conducting a factorial design experiment, rather than examining each factor separately, as a pilot study before initiating a full‐scale noninvasive research project.     

Predator specificity of kairomones in diel vertical migration of Daphnia: a chemical approach

Daphnia responds to chemical cues released by fish with diel vertical migration (DVM) as a behavioural predator avoidance. We used a bioassay to characterize the chemical nature of the kairomone. Cues released from stickleback ( Gasterosteus aculeatus , Gasterosteidae) and a piscivorous pike ( Esox lucius , Esocidae) were enriched from incubation water by reversed-phase sorbent extraction and were reversibly inactivated by acetylation. HPLC yielded only one active fraction with identical retention times for the kairomones of both species. Chemical features did not differ from those previously reported for Cyprinidae, indicating that the active compounds are very similar if not identical. From further investigation of the kairomone released by crucian carp ( Carassius carassius , Cyprinidae), glucuronic acids and carboxy-, sulphate- and phosphate-groups can be excluded as essential for biological activity. The response of Daphnia increased with increasing concentrations of extracted kairomone. The kairomone was not released from mucus by digestion with hyaluronidase. Adsorption of the kairomone to food particles seems to be of minor importance suggesting that the cue is perceived as a freely dissolved molecule.     

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.     

A refined blood collection method for quantifying corticosterone

In many rodent laboratories, blood samples are collected from rats using the tail vein nick procedure and analyzed to quantify blood corticosterone levels as an indicator of stress. The standard method of corticosterone quantification often requires the collection of a relatively large volume of blood, followed by the extraction of the blood plasma. An alternative blood sampling method requires the collection of only a drop of blood on paper (the 'drop' method), minimizing handling of the animals, and does not require plasma extraction. The authors aimed to validate the drop method of blood sampling for use in corticosterone quantification. They induced stress in rats by cerebral ischemia, collected blood samples at various intervals using both the drop method and the plasma extraction method and then quantified corticosterone by radioimmunoassay. Corticosterone levels of the ischemic rats were compared with those of sham-operated rats and those of ischemic rats that had been given metyrapone, a glucocorticoid synthesis inhibitor, prior to vessel occlusion. Blood corticosterone levels in the samples obtained from the same animal using the two different methods were highly correlated for all rats. The authors further provide a regression model that can be used to predict plasma corticosterone values from those obtained from the drop blood samples. Quantification of corticosterone from only a small drop of blood has many practical and ethical advantages and should be considered as an alternative to standard methods.     

A method for extraction and analysis of high quality genomic DNA from ixodid ticks

Currently, no published methods describe the extraction of high molecular weight genomic DNA from ixodid ticks (Acari: Ixodidae) and commonly used methods of extraction are not well adapted for use with members of this family. A method for extraction of minimally degraded genomic DNA from ixodid ticks that can be completed in one or two days is described. The method produces DNA which is of sufficient size (>24 kb) for use in Southern analysis and which is readily digestible by restriction endonucleases. Southern analysis using a cytochrome P450 gene probe, demonstrates the success of our method with genomic DNA extracted from two species of Ixodidae, the lone star tick, Amblyomma americanum (Linnaeus) and the cattle fever tick, Boophilus microplus (Canestrini).     

Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing

The gastrointestinal tract contains a vast community of microbes that to this day remain largely unculturable, making studies in this area challenging. With the newly affordable advanced sequencing technology, important breakthroughs in this exciting field are now possible. However, standardized methods of sample collection, handling, and DNA extraction have yet to be determined. To help address this, we investigated the use of 5 common DNA extraction methods on fecal samples from 5 different species. Our data show that the method of DNA extraction impacts DNA concentration and purity, successful NGS amplification, and influences microbial communities seen in NGS output dependent on the species of fecal sample and the DNA extraction method used. These data highlight the importance of careful consideration of DNA extraction method used when designing and interpreting data from cross species studies.     

Is there any evidence that aphid alarm pheromones work as prey and host finding kairomones for natural enemies?

1. The aphid alarm pheromone (E)‐β‐farnesene (EBF) is often considered to be used by natural enemies as a prey/host finding kairomone. However, studies show opposing results, some appear to confirm an attraction of aphid natural enemies by EBF whereas others do not provide any evidence for the kairomone function of EBF. 2. To clarify if aphid natural enemies are attracted by the amounts of EBF naturally emitted by aphids, the existing literature was reviewed about EBF attractiveness to aphid natural enemies with consideration of the amounts of EBF used in the studies. 3. Thirty‐one publications that investigated the ability of EBF, aphid cornicle secretion, and attacked aphids, to attract aphid natural enemies were found. Several studies showed an attraction by EBF, but these used much higher amounts of EBF than usually emitted by aphids during a predator attack. Studies investigating EBF amounts similar to what is emitted by aphids are rare and failed to show attraction. Only two studies document an attraction of natural enemies by attacked aphids. 4. As EBF is emitted in very low amounts, not very stable, and only present after an attack, we suggest that aphid‐derived EBF is not a suitable kairomone for most natural enemy species, especially when they are able to use alternative cues. As EBF, amongst other volatiles, is also emitted by herbivore‐induced plants, we propose that natural enemies might use plant‐derived EBF as a synomone to identify aphid‐infested plants via an altered plant volatile bouquet.     

Comparative Examination of Capercaillie (Tetrao urogallus L.) Behaviour Responses and Semen Quality to Two Methods of Semen Collection

Artificial insemination (AI) is very helpful in solving the reproductive and biodiversity problems observed in small, closed avian populations. The successful production of fertilized eggs using AI is dependent on the collection of good quality semen. Two methods of male sexual stimulation and semen collection from captive kept capercaillie (Tetrao urogallus L.), one of the most seriously endangered grouse species in Europe, are compared in this study. Ejaculates were obtained either with the use of a dummy female or by the dorso-abdominal massage method. Differences in the individual responses of the males to the two methods of semen collection as well as in their semen quality were noted. Only sperm concentration (432.4 x 10⁶ mL^-1 with dummy female and 614.5 x 10⁶ mL^-1 for massage method) was significantly affected by capercaillie stimulation method. Sperm motility and morphology were not affected (P≥0.05). Thus, for semen collection from captive kept capercaillie both methods can be used successfully. The dummy female can be an alternative to dorso-abdominal massage method, commonly used for semen collection from domesticated bird species.     

Volatile spider-mite pheromone and host-plant kairomone, involved in spaced-out gregariousness in the spider mite Tetranychus urticae

ABSTRACT. From the host plant-spider mite complex Phaseolus lunatus—Tetranychus urticae Koch a volatile chemical is emitted that acts as a kairomone for the predatory mite Phytoseiulus persimilis Athias-Henriot (Sabelis et al. , 1984a). This kairomone is apparently a byproduct of a vital physiological process and/or it has a function in the biology of the spider mite as well.
The spider mite—host plant complex also emits a volatile spider-mite dispersing pheromone. This is shown in the present study where spider mites were introduced into an odour patch on a horizontal screen in a vertical airflow olfactometer. When spider-mite infested leaves of Lima bean are offered, the spider mites walk mainly straight and soon reach the edge of the screen. On the other hand, when clean Lima bean leaves are offered, the mites walk tortuously most of the time and reach the edge of the screen much later. Artificially damaged plants elicit the same response as undamaged plants. Differences in spider-mite behaviour are observed in the vertical airflow olfactometer when odour of either clean or spider-mite infested leaves is offered. A comparison of the behaviour in these two situations with that when no odour was offered suggests that Lima bean leaves emit a volatile kairomone that activates T. urticae and makes them return after losing the stimulus. A Y-tube olfactometer experiment confirms the existence of this kairomone.
At a low ratio of dispersing pheromone to plant kairomone, the spider mites behave as if only kairomone is present, walking mainly tortuously. At a high ratio they disperse. No aggregation-pheromonal effect is observed.
The possibility that the spider-mite dispersing pheromone acts as a kairomone for P. persimilis is discussed.