Name: application of two-spotted spider mite tetranychus urticae for plant-pest interaction studies
Uploaded: 2014-07-04T13:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies at JoVE.com

This project was funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-046), and Ontario Research Fund&#8211;Global Leadership in Genomics and Life Sciences GL2-01-035 (to M.G. and V.G.). T.V.L. is a postdoctoral fellow of the Fund for Scientific Research Flanders (FWO).

1，维护了叶螨种群的注:蜘蛛螨饲养加州红芸豆（ 菜豆 ）。 <ol><li>种植豆类植物的种子从2-3周前为患。 </li><li>与受侵染植物混用这些植物;成螨会迅速拓殖新鲜的植物材料。 </li><li>删除旧的侵染豆科植物每隔7-10天，用新鲜的植物取代。 </li></ol> 2，收集成年女性螨<ol><li>使用洗涤方法收集螨<ol><li>为了收集螨虫，放置一个新鲜的豆科植物与植物侵染接触实验前1〜2天。 </li><li>用20〜30鲜豆类植物，收集1000〜2000成年女性螨。 </li><li>用自来水在RT制备吐温20的溶液中于0.001％。 </li><li>在吐温20溶液清洗出没的豆类植物，使用2〜3个工厂的时间。 </li><li&gt;每个工厂洗涤2〜3倍，以收集所有叶螨。 </li><li>完成步骤2.1.4在10分钟内，以避免螨死亡率。 </li></ol></li><li>成年女性螨分离的螨过滤悬浮液通过定义网格大小筛<ol><li>准备以下网目尺寸的筛子:500微米（以去除碎片）和300微米（收集雌性成年螨）（ 图1A）。 </li><li>通过500微米的筛子过滤悬浮液。 </li><li>通过300微米的筛子重新筛选暂停。成年女性螨虫将被保留。 </li><li>沾了300微米筛成年女性螨在干净的自来水，除去吐温20。 </li><li>在上面的筛子的底部单层散布螨。 </li><li>用纸巾从筛的网眼和侧面除去过量的水。注意:这样做是因为螨虫需要，以恢复迅速干透即可。 </li><li>准备一个组件， 如图1所示乙;这将允许螨自由移动，但会阻止他们逃跑。 </li><li>将筛上，是用来作为支撑筛的顶螨。 </li><li>环绕声底筛与水，以防止螨虫从分散。注:螨将开始恢复后约5分钟。后约30分钟，移动螨虫的数量是足以启动集合（ 图1D）。螨应在1小时内被收集，因为它们产生的丝，将与集合中产生干扰。 </li></ol></li></ol> 3，植物侵染螨注意:一旦成年雌性螨已痊愈，它们可用于植物侵染。有两种方法用来侵扰实验工厂与成年女性螨:a）使用细刷（协议第3.1节）和b）用泵或真空线（协议第3.2节）。 <ol><li>螨虫为患使用Brush 注:红蜘蛛为害用刷子被用于应用程序的每株最高30螨。 <ol><li>使用大小00或更细的软毛圆艺术画笔。 </li><li>湿刷R​​T自来水。 </li><li>轻轻地捡起筛螨使用画笔笔尖，并将它们传送到工厂。 </li></ol></li><li>用泵螨感染 注意:植物侵染由泵/真空线螨收集时，使用超过30个螨虫被应用。对于这种方法，可以用一个真空泵用于微电子元件拾取或真空管线。它保持恒定气流具有足够的强度和（2-4磅）是重要的。 <ol><li>附1毫升移液管尖，以连接到泵或真空线用1.5ml的离心管中切的底部作为泵/真空管和所述顶端之间的适配器管中。 </li><li>将枪头和适配器（1.5毫升离心管）之间的一块纸巾。注:这样做的目的是为了里面吨螨虫陷阱他的枪头在抽吸过程，同时也减少了气流（ 图1C）。 </li><li>直接使用枪头筛收集螨。 </li><li>另外，从使用立体镜感染的叶子收集螨一个接一个。 </li><li>当已收集螨所需数量的，从管取出枪头，确保了一块纸巾在移液管尖的背面原状。 </li><li>通过点击枪头和结块的螨虫一起收集螨。然后，将它们放置在叶片上;几秒钟内，螨虫会开始驱散在叶片上。 </li></ol></li></ol> 4，记录和评估植物损伤<ol><li>录影厂损伤 注:对于A。拟南芥 ，厂房损坏，经过4天的螨虫喂养评估。损坏记录为褪绿斑的总表面积。对于损坏的测量，整个莲座被切割和扫描。描述扫描参数是一个爱普生V30扫描仪，但将作为一个很好的起点，任何类似的平板扫描仪。保留所有实验的扫描参数不变，允许个别实验运行之间的比较。 <ol><li>覆盖扫描仪床上，透明片，以防止污染扫描器表面的植物材料。 </li><li>切割整个莲座和地点上的扫描仪床上所以叶子的正面的（上）侧朝向扫描仪的光源和捕获元件。另外，如果花环过于密集捕获单个叶片不重叠，解剖莲座成单个叶片或不重叠的叶子用细剪刀组放置在扫描仪上床前。多个莲座可以同时被扫描。 </li><li>覆盖玫瑰花或树叶用一张白纸，以防止遵守和扫描器盖板的污染。 </li><li>关闭扫描仪盖板，并以下列参数进行扫描分辨率:1,200 DPI;色彩模式:Adobe RGB的;亮度:25;文件类型:最高质量JPEG格式。 </li><li>保存图像文件，用于后续的损害分析。 </li></ol></li><li>对植物的损伤定量 注:损伤的面积是使用Photoshop手工计算。由于叶片的形状和症状的颜色的强度作为螨馈送的结果，大的差异，自动的方法是不可靠的。因此，网格的方法是用来评估对植物的损伤: <ol><li>打开工厂用Photoshop的图像。 </li><li>创建一个新层，然后覆盖扫描图像为0.25 mm×0.25毫米区划网格（视图或显示 - 显示 - 网格，键盘快捷键Ctrl +&#39;）。 </li><li>在一个覆盖层，利用标记点（使用"铅笔工具"，快捷键B）损坏叶面积。用于网格下面这有损坏覆盖超过一半的网格单元中的每方一一个点。点的大小是在像素定义（ 即 </eM&gt;每点52像素）。 </li><li>当电网中所有的广场，展示植物的伤害，涵盖至少有一半的单元格中都标有一个圆点，使用直方图工具（窗口 - 直方图），以确定该层上的像素的总数。直方图工具允许一个测量的像素的数目。由于每一个点是由像素的恒定和已知的数来表示，直方图工具将测量的总像素数，代表点的总数，并且，通过扩展，标志着正方形的总数。 </li><li>标计算使用公式"点"的数量: 点阵数=每点像素的像素/数总数。 </li><li>通过1平方网格的区域在图像上标记相乘的点的数量，作为一个点对应于电网的1平方计算损害的总面积。 </li></ol></li></ol>

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The authors have nothing to disclose.

该视频演示了用于分离和侵扰的植物有大量的成年女性螨的协议。虽然我们使用甲。此协议芥 ，它可用于任何植物的叶螨相互作用系统，目前正在顺利也适用于番茄和葡萄（ 葡萄 ）的植物。该协议的产量可再现的结果，表明收集的螨虫是可比的生理状态（ 图3）。 虽然这些协议是简单的执行，有几个关键的步骤需要特别注意，因为它们会影响螨恢复。从叶螨洗衣机必须达到三豆工厂同时进行，并具有在10分钟内利用水在室温下完成。此外，螨必须被传播的筛子进行快速干燥上。如果螨恢复不理想，它检查的吐温20洗涤剂的推荐量是否是我们很重要ED和如果洗涤剂在漂洗步骤期间完全移除。 有两个主要限制到协议提出:1）螨收集由泵可以很好地用于超过30螨但收集的螨虫的数量较少，需要使用一个刷子和相对缓慢的集合;然而，收集并通过洗涤方法集中成螨大大方便了他们的应用程序;二）损伤分析，通过打分褪绿斑点表面可有时间和精力耗费步骤;今后的努力应该躺在识别，可以用来作为螨饲养的测量标记基因。 相比以前发表的方法，红蜘蛛集合的呈现方法提供采集大量可行的个人和发育阶段在一个步骤中有效地分离的优点。此外，机器损坏的表型在植物病虫害的相互作用，通过视觉inspecti评估上或数字图像分析通常是一个复杂的分析，涉及某种形式的分子读出（如基因表达分析或代谢物图谱）的第一步骤。目前的协议是适用于少数螨和收集，这对于植物和害虫之间的长期相互作用的分析有价值的材料天后出没的应用;然而，该协议使一个同时适用于数百螨有效地捕捉在更短的时间内（小时），并在觅食地发生的植物反应。 综上所述，申请成年女性螨对寄主植物和植物的损害评估方法进行了描述。这些协议是对实验旨在了解植物和二斑叶螨的相互作用的遗传和分子基础是必不可少的。

植物病虫害的相互作用具有重要的科学和经济重要性的话题。它使用两作物植物（如番茄）和模式植物，A.历史研究拟南芥 。在这两种情况下，植物虫害的易感性可以是食草动物后发作或间接地通过对害虫表现的评估测量直接通过植物的表型评估。 植物敏感性的直接测量以前用于使用各种方法的一些害虫种类。例如，鳞翅目幼虫取食被测量为所消耗的任小菜蛾 （小菜蛾）或粉纹夜蛾 （卷心菜尺蠖），用肉眼与电网12的帮助下植物组织的部分的估计。还有，也有利用的叶损害与随后的定量图像分析的数字成像方法。这种方法是在使用A.研究与西花蓟马 （西花蓟马）13，Scaptomyza黄花 （叶采果蝇）14和T拟南芥互动妮 15。 植物易感性间接测量被广泛应用于植物病虫害的互动研究。例如，易感A的拟南芥对桃蚜桃蚜取食通常是通过害虫产卵量和互动16,17后植物的大体形态的描述，分析评估。 A的另一个典型的间接指标拟南芥敏感性害虫是草食动物的干或湿重评估。这个参数是常用来表征取食lepidoterans，如菜粉蝶 （白色小），P.小菜蛾 ，或T。妮在其幼虫或蛹的阶段15,17。 红蜘蛛是细胞铁含量eders。螨虫引起的损伤是公认的褪绿斑点，颜色从白色到浅绿色的集合。植物宿主红蜘蛛虫食的易感性先前评估或者间接地通过对红蜘蛛性能日内进行分析后虫蛀18,19，或直接使用的植物星期后虫蛀18的大体形态，或使用数字树叶的成像曝光几天螨与随后的自动图像分析19。这些方法被开发并用于番茄植株和T之间的相互作用的研究螨，和通常使用少量的蜘蛛螨（5-15每处理）从混合螨种群收集和使用软毛刷被放置在叶片表面上。然而，这些方法不适合于研究，其中需要更大数目的螨虫被应用。此外，在叶片图像直接处理在图像分析软件如Adobe Photoshop（圣何塞，加利福尼亚州）的ImageJ或20可用于番茄损伤分析中，这些协议需要修改以适用于具有表面反射率大于或浅色的并且具有高度可见的毛状体（叶片例如 ， 拟南芥 ）上自动选择褪绿斑，标志着植物伤害干扰。此外，可与以前的方法很容易地利用蜘蛛螨的发育阶段被限制在最普遍的和易于识别的成年女性和排除利用其他发育阶段。 对植物叶螨相互作用的高通量分析的第一个关键步骤是建立可重复的，简单的和可靠的协议，挑战与植物叶螨和可靠的评估互动的结果。 在这段视频中，可快速方便地收集大量图片的有效方法成年女性螨的误码率，他们的实验植物宿主和植物造成的损害红蜘蛛喂养评估上的应用进行了描述。所提出的协议可实现快速和有效地收集数百人在任何发育阶段（卵，幼虫，若虫，成年男性和女性），可以用于后续的实验应用。此外，这些协议可以被应用到任何螨宿主植物，但在A的情况下是特别展示拟南芥 。

<table><tbody><tr><td>Plant material:</td><td></td><td></td><td></td></tr><tr><td>California red kidney bean</td><td>Stokes, Thorold, ON, Canada</td><td></td><td>2 week old, well infested with spider mites 2 or 3 days before use. Other cultivars of &lt;em&gt;Phaseolus vulgaris&lt;/em&gt; can be used.</td></tr><tr><td>Chemicals:</td><td></td><td></td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P9416</td><td>1% stock solution is prepared to simplify aliquoting</td></tr><tr><td>Tap water</td><td></td><td></td><td>At room temperature, heat- and cold-shock affect mite survival rate and performance</td></tr><tr><td>Other materials and equipment:</td><td></td><td></td><td></td></tr><tr><td>Plastic tray</td><td></td><td></td><td></td></tr><tr><td>Set of scissors</td><td></td><td></td><td></td></tr><tr><td>2 L beakers</td><td></td><td></td><td></td></tr><tr><td>Paper towels</td><td></td><td></td><td></td></tr><tr><td>Sets of sieves</td><td>Manufactured in house</td><td></td><td>Detailed instructions are available</td></tr><tr><td>Thin brush</td><td></td><td></td><td></td></tr><tr><td>Pipettes</td><td></td><td></td><td></td></tr><tr><td>Pipette tips</td><td></td><td></td><td>0.2 and 1 ml</td></tr><tr><td>1.5 ml centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>Air pump</td><td></td><td></td><td>Aquarium type pump with inverted air flow. Vacuum line can be used. Required pressure drop is approx. 2-4 psi</td></tr><tr><td>Stereoscope</td><td></td><td></td><td></td></tr><tr><td>Scanner</td><td>Epson</td><td>V30</td><td>Any flatbed scanner allowing necessary degree of control over scan quality. We use Epson V30 for our experiments.</td></tr><tr><td>Computer</td><td></td><td></td><td>Windows or OS X PC which is compatible with scanner hardware and Adobe Photoshop software.</td></tr><tr><td>Adobe Photoshop software</td><td>Adobe Systems Inc., San Jose, CA, USA</td><td>various</td><td>Any version with Histogram tool included.</td></tr></tbody></table>

application of two-spotted spider mite tetranychus urticae for plant-pest interaction studies

二斑叶螨， 二斑叶螨 ，是一种普遍存在的多食性节肢动物食草动物，饲料是一个非常广泛的物种阵列，拥有超过150经济价值上。它是温室作物的主要害虫，特别是在茄科和葫芦科 （ 例如 ，西红柿，茄子，辣椒，黄瓜，西葫芦）和温室观赏植物（ 如玫瑰，菊花，康乃馨），年大田作物（如玉米，棉花，大豆，糖用甜菜），和多年生栽培（苜蓿，草莓，葡萄，柑橘，和李子）1,2。除了 ​​极端的polyphagy，使得它的一个重要农业害虫，T。螨有一种倾向，产生耐药性了用于其控制3-7杀虫剂和杀螨剂的广泛。 T.螨是一个很好的实验生物，因为它有一个快速的生命周期（7天，27°C）并且能够容易地维持在高密度在实验室。的方法来测定基因表达（包括原位杂交和抗体染色）和失活的利用RNA干扰红蜘蛛内源基因的表达已开发8-10。近日，T的全基因组序列螨有报道，创造了一个机会发展这种害虫草食动物作为模式生物与已经在一些寄主植物（ 拟南芥和番茄茄lycopersicum）11的存在相当于基因组资源。总之，这些模式生物可以提供深入了解植物害虫相互作用的分子基础。 在这里，可快速方便地收集了大量的成年女性螨，他们的实验厂主机上的应用程序，和植物造成的损害红蜘蛛喂养评估的有效方法进行了描述。所提出的协议连接埃布尔斯快速和有效地收集数百人在任何发育阶段（卵，幼虫，若虫，成年男性和女性），可以用于后续的实验应用。

用20〜30出没的豆科植物，可以用筛子收集约2,000成年女性螨。侵扰10个工厂，每个工厂20螨所需要的时间大约是15分钟，如果用刷子转移螨虫。的收集和应用方法的组合示于图2。 该协议产生的植物的损害可重复的结果，表明收集的螨虫是相似的生理状态，适合于植物叶螨相互作用研究（ 图3 A）。评估协议的再现性，使用的洗涤方法，其中，20螨置于3周龄A.莲座叶收集成年雌性螨拟南芥植物（加入Col-0中），用湿刷子。使用上面描述的技术处理的植物被扫描4天postinfestation和损伤的面积进行定量。做用单因素方差分析重复实验结果的比较。由于实验应用的一个例子，我们评估的敏感性自然变异红蜘蛛虫食在3个A。用湿刷子和记录伤害4天后侵染拟南芥种质通过应用每株20母蜘蛛螨。在扫描的图像损坏的典型外观示于图3B中 。随后，破坏数据的量化可以表示为条形图或箱线图，并通过所选择的统计方法（ 图3C）进行分析。在这个例子中，ANOVA，随后由杜克HSD检验进行数据分析。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51738/51738fig1highres.jpg" width="500" /> 图1中，实验设置为成年雌性螨的隔离。 （一）设置筛子，允许不同的叶螨发育的分离阶段。 （A.0.5毫米开孔尺寸 - 从螨悬挂杂物去除B级0.3毫米 - 。收集成年女性螨C 0.2毫米 - 。收集年轻女性，男性和若虫ð0.16毫米 - 收集幼虫和若虫É。 0.1毫米-用来让螨虫无洗涤和过滤后逃逸恢复收集鸡蛋）（B）设置（C）设置用于通过泵收集螨（D）筛与移动成年女性螨准备收集。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51738/51738fig2highres.jpg" width="500" /> 图2，可能的策略，以隔离成年雌性螨，根据螨申请的编号，如果实验设计需要使用大量的成年雌性蜘蛛螨，最有效的方法是直接使用真空泵收集方法从豆叶。如果实验需要使用一个发育阶段比成年女性或少数个体需要被小心地放在年轻植物其他的，则建议使用分子筛随后洗涤的方法进行收集的叶螨的发育阶段和分馏虫蛀用刷子。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51738/51738fig3highres.jpg" width="500" /> 图3代表性结果。 （一）重复性的实验结果。 3周龄A.测量损伤面积拟南芥植物，加入Col-0中，应用20母蜘蛛螨后4天。做重复实验结果的比较采用单因素方差分析（N = 6，F = 0.621，P = 0.735）。跨越3（B和C）自然变异易患红蜘蛛虫食拟南芥加入的国家:Col-0中，LER-0，和WS2（B）外观植物的叶螨的取食后。红色箭头指向的典型破坏的地区。（C）差异易感性红蜘蛛虫食所评定损伤的区域。易患红蜘蛛虫食跨种质显著变化（方差分析中，n = 6，F = 13.4，P = 0.0004）。字母表示在P &lt;0.05（图基HSD检验）基因型之间的显著差异。作图的值是平均值的平均值±标准误差。

Watch this Scientific Journal Video about Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies at JoVE.com

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

协议对红蜘蛛，实验植物的侵染和植物的损害评估的均匀样品的制备效率的要求，对植物病虫害的相互作用研究，开发。

二斑叶螨的应用<em&gt;二斑叶螨</em&gt;植物病虫害的相互作用研究

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with ...

application-two-spotted-spider-mite-tetranychus-urticae-for-plant

Nanyang Technological University

Research

JoVE Journal

Environment

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

17.6K 次观看.  The University of Western Ontario. 协议对红蜘蛛，实验植物的侵染和植物的损害评估的均匀样品的制备效率的要求，对植物病虫害的相互作用研究，开发。 

视频: Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

Remote Sensing Evaluation of Two-spotted Spider Mite Damage on Greenhouse Cotton

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the two-spotted spider mite (TSSM), Tetranychus urticae Koch, on greenhouse grown cotton. TSSM is a polyphagous pest which occurs on a variety of field and horticultural crops. It often becomes an early season pest of cotton in damaging proportions as opposed to being a late season innocuous pest in the mid-southern United States. Evaluation of acaricides is important for maintaining the efficacy of and preventing resistance to the currently available arsenal of chemicals and newly developed control agents. Enumeration of spider mites for efficacy evaluations is laborious and time consuming. Therefore, subjective visual damage rating is commonly used to assess density of spider mites. The NDVI (Normalized Difference Vegetation Index) is the most widely used statistic to describe the spectral reflectance characteristics of vegetation canopy to assess plant stress and health consequent to spider mite infestations. Results demonstrated that a multispectral optical sensor is an effective tool in distinguishing varying levels of infestation caused by T. urticae on early season cotton. This remote sensing technique may be used in lieu of a visual rating to evaluate insecticide treatments.

This manuscript describes a multispectral optical sensor that effectively detected damage to early season cotton artificially infested with varying densities of the two-spotted spider mite populations.

温室棉二斑叶螨危害的遥感评价

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the ...

科研

环境科学

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

的发展<em&gt; Metarhizium anisopliae</em&gt;作为防腐剂:从隔离到现场表现

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

分离叶测定,以简化马铃薯中基因表达研究,在咀嚼昆虫曼杜卡性塔感染

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

生物化学

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides in attempts to control its populations eliminates natural enemies and competitor flower thrips species, thereby increasing its populations. An unsustainable situation develops with concomitant resistant pest populations, secondary pest outbreaks, and environmental degradation. Integrated pest management utilizes knowledge of pest and natural enemy relationships to implement tactics that are environmentally friendly and sustainable. Minute pirate bugs are the most important worldwide predators of thrips. They can suppress and ultimately control Frankliniella species flower thrips. Flower samples taken at least weekly are needed to understand predator-prey dynamics. Shown here is the sampling of the flowers of fruiting vegetables and companion plants to estimate the densities of individual thrips and minute pirate bug species. Representative data illustrates how the protocol is used to determine the efficacy of management tactics over time and how to evaluate the benefits of predation by minute pirate bugs. The sampling protocol is similarly adaptable to sampling thrips and minute pirate bugs in other plant species hosts.

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments

Presented here is a protocol to determine the number of thrips and minute pirate bug predators in crops over multiple dates in field experiments. Also illustrated is how to determine the efficacy of management tactics against thrips and evaluate the benefits of predation by minute pirate bugs.

现场实验中估计富兰克林菌种花纹和奥里乌斯物种捕食者的取样

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides ...

Leveraging Micro-CT Scanning to Analyze Parasitic Plant-Host Interactions

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility of three-dimensional visualization and virtual sectioning, has allowed novel and increasingly detailed analysis of complex plant organs. Interactions among plants, including between parasitic plants and their hosts, can also be explored. However, sample preparation before scanning becomes crucial due to the interaction between these plants, which often differ in tissue organization and composition. Furthermore, the broad diversity of parasitic flowering plants, ranging from highly reduced vegetative bodies to trees, herbs, and shrubs, must be considered during the sampling, treatment, and preparation of parasite-host material. Here two different approaches are described for introducing contrast solutions into the parasite and/or host plants, focusing on analyzing the haustorium. This organ promotes connection and communication between the two plants. Following a simple approach, details of haustorium tissue organization can be explored three-dimensionally, as shown here for&#160;euphytoid, vine, and mistletoe parasitic species. Selecting specific contrasting agents and application approaches also allow detailed observation of endoparasite spread within the host body and detection of direct vessel-to-vessel connection between parasite and host, as shown here for an obligate root parasite. Thus, the protocol discussed here can be applied to the broad diversity of parasitic flowering plants to advance the understanding of their development, structure, and functioning.

Micro-CT is a non-destructive tool that can analyze plant structures in three dimensions. The present protocol describes the sample preparation to leverage micro-CT to analyze parasitic plant structure and function. Different species are used to highlight the advantages of this method when coupled with specific preparations.

利用显微CT扫描分析寄生植物与宿主的相互作用

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

生物测定对杀虫剂的抗药性监测

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

生物学

Trichogramma dendrolimi 和毒液提取的人工宿主寄生

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

作者聚焦：Trichogramma 寄生黄蜂的高效毒液提取方法 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

二斑叶螨的应用

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从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

现场实验中估计富兰克林菌种花纹和奥里乌斯物种捕食者的取样

利用显微CT扫描分析寄生植物与宿主的相互作用

生物测定对杀虫剂的抗药性监测

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液