Name: bioassays for monitoring insecticide resistance
Uploaded: 2010-12-30T16:30:00
Duration: 6 min 30 s
Description: Watch this Scientific Journal Video about Bioassays for Monitoring Insecticide Resistance at JoVE.com

我们想感谢资助研究有关本刊物的美国棉花公司的国家支持计划＃08 317MO，美国农业部/ CSREES SR - IPM的格兰特2009-34103-20018和兰迪斯国际。

1。简介已成为与实现最重要的粮食生产，到2025年，世界人口将达到8.04亿人 1 。届时将有需要提供数额较大的粮食作物，比目前生产。充足的粮食供应不会没有如农药等植物保护产品的使用能够增加作物产量的数量和质量保持。基于对农药的历史和当前的依赖，抗药性的情况会继续发生，在科学文献中报道。 抗虫性农药是有问题的，因为产品是一个不可分割的高产和高品质的生产的农业组成部分。然而，农药的过度使用和/或滥用可导致发展的阻力，这可能不利于作物生产。害虫（即，虫，杂草，病菌等），通过各种机制，但发展的阻力主要驱动因素是缺乏登记的农药1可用的独立行动模式发展的阻力。当个别害虫标记在一个特定的作物系统的少数产品，化学品控制选项是有限的。因此，相同的化学（S）是反复使用，不断的选择压力是害虫。这个问题加剧时，害虫已在一年多代，每一代人接触到农药。 有财政和enrivornmental相关的成本与阻力2。抗虫性会导致更高的利率和更频繁的应用程序所需的农药，以达到满意的控制。产量损失可能增加，因为未能控制目标害虫的农药使用后，即使发生。皮门特尔2抗药性这些费用估计在美国每年约15亿美元。 农药害虫种群之间的易感性调查是主动的方式来检测任何杀虫剂性能的转变，并提供预警，修改化学控制策略。通过修改整体的IPM战略，对一个给定的农药的可行性可能被延长，这反过来，重要的是为农业，继续为世界提供足够的食物和纤维。本文将描述一个可以用来监控杀虫剂的易感性，并检测出抗杀虫剂的接触杀虫剂的使用成人小瓶测试3的人口发展和修改这个测试厂房4系统性杀虫剂的协议。 2。方法 2.1安全声明用杀虫剂处理时，使用适当的安全预防措施是非常重要的。咨询适当的个人防护设备（PPE），前处理农药的材料安全数据表（MSDS）。应要求处理农药在实验室特定的实验室培训的人员，然​​后再尝试任何生物测定。 2.2的股份方案制定一个从已知浓度的杀虫剂的活性成分（AI）的源的原液。所有所需的浓度，包括在生物活性的治疗将会从原来的原液。对于本文中，将发展作为原液100微克/ ml的溶液100mls的利益，但是，任何浓度可以从初始浓度稀释。调整的技术等级的AI％纯度的基础上加以权衡。 权衡金额=（体积使）×（[＃微克/ ml的溶液] / [％purity/100]） 例如，它需要的技术人工智能10152.284微克到100微克/毫升的98.5％纯度的技术等级杀虫剂溶液100毫升： （100毫升）×（100μg/ml] / 0.985）= 10152.284微克 2.2.1成人小瓶联系杀虫剂试验部分填补容量瓶中，用丙酮和杀虫剂AI权衡调整后的数额。冲洗烧瓶中，用丙酮重量船，删除权衡船所有杀虫剂。填写的容量瓶中，以用丙酮毕业线。在冷藏条件下举行的大多数解决方案，将保持与疗效没有明显的损失约一个月的活动。 2.2.2成人系统性杀虫剂瓶测试的修改昆虫与系统性杀虫剂中毒是接触的产品截然不同。全身产品通常必须摄取昆虫的昆虫，而不是成为积极havin克直接接触的产品与接触杀虫剂。因此，成人小瓶测试被修改为了要测试的系统性杀虫剂。原液所需的AI的重量计算在2.2节中提到的类似。的方法是相似的，除了AI在10％重量蜂蜜水溶液溶解。该解决方案应在24小时开始的生物活性。如果技术等级材料是不溶于水，高浓度的杀虫剂解决方案可与丙酮等，添加蜂蜜，只有一个小的杀虫剂量：丙酮：水解决方案 4 。 2.3浓度确定的浓度范围内与发展确定适当的浓度，需要建立生物测定范围的反应会很困难​​。可能需要几个重复定义的浓度范围内，这是通过试验和错误。此外，范围可能随时间而改变，如果人口变化的易感性的水平。许多其他因素，应考虑建立终浓度，包括：家庭昆虫，杀虫剂类，昆虫的大小等，此前公布的成人瓶测试的结果可以帮助杀虫剂浓度的初始选择。已发表 ​​：半翅目：蚜，蝽8-10和11-14盲蝽5-6 Aleyrodiidae; Thysanaptera：蓟马科15;鞘翅目：瓢虫18日至19日和Cybocephalidae 19日17日，16日，象甲科Brentidae;双翅目：蚊的生物测定20;鳞翅目：卷蛾科21日和22日至26日，夜蛾科3;膜翅目：茧蜂科7,18，Aphidiidae 18月26-27姬蜂科，跳小蜂科7，和Aphelinidae 18。 浓度的基础上的“X”微克/ ml的溶液。然而，当瓶成人小瓶测试准备，只0.5毫升将被添加到每个小瓶，因此，小瓶中的浓度一半浓度。一旦浓度选择，下面的公式可以帮助确定解决方案的数量添加到所需浓度。 （C1）（V1）=（C2），（V2） 其中C1是原液的浓度; V1是，使新的浓度所需的库存量; C2正在准备的浓度;，V2是新的浓度量。例如，一个100μg/ml原液5mls需要做出一个5μg/ml100mls。这5μg/ml的解决方案会导致涂在2.5μg/vial浓度的小瓶。 5mls = [（5μg/ml）（100mls）] /（100μg/ml） 2.3.1成人小瓶联系杀虫剂试验部分填补体积flaks，并分装的原液需要作出所需浓度的计算量。用丙酮毕业线填充容量瓶中，并进行第2.4节或密封储存在冰箱以备后用封口膜M（加拿大铝业公司，尼纳，威斯康星）。大多数解决方案都为1个月的良好。 2.3.2成人系统性杀虫剂瓶测试的修改这个过程是相同的，如以前在2.3.1中提到的，除由蜂蜜重量的10％的丙酮水溶液取代。混合足够的蜂蜜：水的解决方案，使所有所需的浓度。这些解决方案应配制后24小时内使用。 2.4准备小瓶 2.4.1成人小瓶联系杀虫剂试验如果浓度分别存放在冰箱里，让温暖的环境温度。基于温度的丙酮，这可能会影响杀虫剂浓度的变化量。颜色代码20毫升玻璃瓶装上螺丝帽浓度使用油漆或标记。 调整重复移液器提供0.5ml/concentration。启动用丙酮控制工作，以下的最低浓度和浓度的增加继续，直到所有的浓度一直使用。实际处理的小瓶（即40瓶20mls）在小烧杯中倒入约一半量的解决方案。的解决方案是在不使用时，盖，以尽量减少蒸发，这可能会导致浓度变化。绘制成重复移液器杀虫剂的解决方案，并免除成单独的玻璃小瓶0.5毫升解决方案。 治疗后，立即玻璃瓶放在商业热狗辊。由于滚筒转，玻璃瓶旋转和丙酮挥发，留下内与技术学校小瓶涂人级杀虫剂。热量可以降低杀虫剂，因此，它是重要的热量被关闭。这可以通过一个模型，加热和辊同时在同一个交换机或使用热狗辊有独立运作的加热元件和滚筒的开关模型控制加热元件断开。避免把更多的小瓶比辊持有;丙酮蒸发前的玻璃小瓶中旋转，他们可能不会收到小瓶墙壁上更涂层。 允许旋转，直到所有的丙酮蒸发的小瓶。可能有小瓶墙壁上一层薄薄的丙酮缩合;因此，小瓶需要单独审理。所需时间推出基于实验室条件下的小瓶不同。一旦小瓶干燥，帽，它们存放在冷冻或暗的条件。已观察到的昆虫休息盖子上低于小瓶唇（即，避免处理过的表面）挂的锥形衬垫或撕裂铝箔内衬，因此，重要的是利用联合国内衬盖。 任何剩余的解决方案，不使用准备小瓶应妥善处置。容量瓶中剩余的解决方案，可以用封口膜密封，存放在冰箱里。日期时，他们准备的小瓶，因为不同的杀虫剂有不同的保质期。例如，菊酯类涂层小瓶约一个月，而涂有一个更不稳定的杀虫剂，如有机磷，小瓶，两周甚至更短的保质期。 2.4.2修改成人系统性杀虫剂瓶测试花卉泡沫作为基材提供杀虫剂的解决方案，以昆虫 4 。剪下使用软木螟的花卉泡沫件（采用12mm × 12mm）。将一块花卉泡沫到先前所描述的的玻璃小瓶。调整，以提供0.5毫升解决方案和分配到花香的泡沫填充重复移液器。这种液体的体积饱和一块花卉泡沫，但不应超过样品瓶中的泡沫水平。同样的工作顺序控制（10％重量蜂蜜水溶液）和低浓度最高。 2.5。存储样品瓶 2.5.1成人小瓶联系杀虫剂试验当存储涂层瓶杀虫剂，杀虫剂知道的属性。不同的杀虫剂有不同的存储要求，如：除虫菊酯光敏感，可存放在室温下，但在黑暗中，但是，有机磷杀虫剂对温度敏感，需要在冷冻储存。如果杀虫剂须存放在冷冻，必须加热到室温之前暴露的昆虫。 2.6生物测定收集的昆虫进行测试。这个过程可以通过使用信息素诱饵，扫网，或任何其他手段大规模捕捉。昆虫应为8 - 24小时，以便发生在收集过程中受伤的人的自然死亡率;他们提供食物和水分的来源，在此期间举行。 10-25％浓度的昆虫（10最低）;测试昆虫数量较大的，更强大的数据集将进行测试。当选择昆虫的生物活性，选择健康，积极的个人和丢弃昏睡或异常的个人。这样，他们接触到全方位的浓度不只是一个时间浓度在小瓶放置昆虫。此过程将防止放置在一个最健康，最活跃的个人或少数浓度。此外，如果它是不可能的至少10％浓度的昆虫进行实验，每个浓度暴露尽可能多的。然后从很短的时间内同一样品区（两三个星期）收集更多的昆虫和重复实验。有些昆虫可以失去本榨季进展的易感性（建设阻力）;因此，在早春收集的个人的反应可能会在秋季28收集的不同。 制定标准，以评估死亡率。最常用的垂死或死亡的昆虫分类的标准是缺乏协调运动。这些意见可能包括权利本身无力如果放在其背水面上，无法保持协调飞行1m或缺乏协调运动时，轻轻地用一个钝器打了招呼。如果个人权利本身，但属于过去，有没有一个协调的运动;昆虫，应视为“死”。昆虫可能会遇到困难，扶正一个光滑的表面上，因此，它也许有必要提供的昆虫的表面，使其能够获得必要的权利本身的牵引。记录幸存者和死者的个人计算每个浓度的存活数量。 2.6.1成人小瓶联系杀虫剂试验放入小瓶昆虫（S）和松散的安全上限。盖子需要，以防止逃逸的昆虫，但松动到足以让空气流通。对于大多数昆虫的生物测定，昆虫只有一个是摆在每一瓶;然而，小昆虫，如粉虱，蓟马，可能会暴露率多达30个， 每 6 瓶，15个人。在室温下放置小瓶直立，直到昆虫生物活性的端点死亡率评估。曝光时间可以有所不同物种和杀虫剂可能3,5-27。在建立一个新的昆虫或化学的初步毒性水平的过程中，监测计划曝光后多个时间点的学科。考试可以终止时浓度最高的死亡率是100％，同时仍保持在非杀虫剂处理过的控制的高存活率（&lt;10％的死亡率）。如果没有控制的高存活率最高浓度达到100％的死亡率是，生物测定应重复使用较高浓度的范围。理想的情况下，死亡率水平应作为杀虫剂浓度的增加而增加。 有些昆虫，暴露24小时，需要的水分来源（即，植物材料的一小片）12 。当一个微妙的昆虫工作，有利于确定昆虫可以在小瓶中生存多久杀虫剂暴露在成人的小瓶测试之前，没有水分的来源。这些信息可以由放置在一个小瓶，没有水分来源的昆虫，再进行实际杀虫剂调查和监测其随着时间的推移生存。 2.6.2修改成人系统性杀虫剂瓶测试在此之前启动的生物测定，确定昆虫饲料和生存与蜂蜜饱和花卉泡沫：在没有杀虫剂水溶液。饱和与蜂蜜：水（或蜂蜜：水的丙酮最大的准备与非水溶性的杀虫技术等级浓度使用量）花卉泡沫瓶放置昆虫和监测存活数天。这种修改成人小瓶测试只审查了一个盲蝽和蝽29;因此，决定在测试期间，需要多接触杀虫剂生物测定实验。例如， 绿盲lineolaris（Palisot Beauvois）在24小时后，接触到噻虫嗪的死亡率进行了评估，但72小时吡虫啉 4 。死亡率Oebalus pugax F.在96小时内评估时，接触到呋虫胺 29 。因此，在控制小瓶昆虫的生存，必须始终如一的高之前，可以作出准确的死亡率评级从杀虫剂中毒了好几天。 饱和花卉泡沫放入闪烁瓶中的昆虫（S）。有盖密封的小瓶中，密封，用棉球的小瓶。在室温下放置小瓶直立，直到昆虫的死亡率评估。如前所述，定期率死亡率。考试时有100％的死亡率与控制的高存活率（&lt;10％的死亡率）的最高浓度，可以终止。它可能是必要的测试额外的浓度。 2.6.3数据分析正确的使用一个公式根据雅培30日在控制治疗的死亡率。 校正死亡率（％）=（（％存活控制 - ％生存对待）/％存活控制）× 100 使用日志剂量概率分析数据，确定，杀死人口的50％（LC 50），并建立95％置信区间（CL）的致死浓度。多个软件程序来确定的LC 50（SAS PROC PROBIT 31，马球加32）。 3。注释<ol><li>工作与负流量空气罩下的丙酮和杀虫剂。 </li><li>灌装容量瓶时，最好是有两个丙酮洗瓶子。洗瓶，已为适应快速交货的液体和传递速度缓慢。删除交付手臂的一部分，以创造一个更大的开放，这瓶可以迅速的一个大批量交付使用。一旦丙酮临近毕业线，改变瓶，洗瓶慢交付使用，让你有更多的控制，配发金额。如果一个烧瓶中充满毕业线以上，离开顶部关闭，使丙酮挥发到毕业线。&lt;/ LI&gt; <li>注意股票的解决方案所需的金额，需要准备的浓度，即，如果您填写烧瓶¾的方式，但需要添加30mls，您可能会超过毕业线。 </li></ol>

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M., Leonard, B. R., Cook, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+and+field+evaluations+of+insecticide+toxicity+to+stink+bugs+(Heteroptera+Pentatomidae).">Laboratory and field evaluations of insecticide toxicity to stink bugs (Heteroptera Pentatomidae).</a> J. Cotton Sci. 7, 156-163 (2003).</li><li>Snodgrass, G. L., Adamczyk, J., Gore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+insecticides+in+a+glass-vial+bioassay+to+adult+brown,+green,+and+southern+green+stink+bugs+(Heteroptera:+Pentatomidae).">Toxicity of insecticides in a glass-vial bioassay to adult brown, green, and southern green stink bugs (Heteroptera: Pentatomidae).</a> J. Econ. Entomol. 98, 177-181 (2005).</li><li>Nielsen, A. L., Shearer, P. W., Hamilton, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+insecticides+to+Halyomorpha+halys+(Hemiptera:+Pentatomidae)+using+glass-vial+bioassays.">Toxicity of insecticides to Halyomorpha halys (Hemiptera: Pentatomidae) using glass-vial bioassays.</a> J. Econ. Entomol. 101, 1439-1442 (2008).</li><li>Dennehy, T. J., Russell, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+Lygus+bug+populations+in+Arizona+to+acephate+(Orthene)+and+bifenthrin+(Capture)+with+related+contrasts+of+other+insecticides.">Susceptibility of Lygus bug populations in Arizona to acephate (Orthene) and bifenthrin (Capture) with related contrasts of other insecticides.</a> Proceedings Beltwide Cotton Conferences. , (1996).</li><li>Snodgrass, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass-vial+bioassay+to+estimate+insecticide+resistance+in+adult+tarnished+plant+bugs.">Glass-vial bioassay to estimate insecticide resistance in adult tarnished plant bugs.</a> Heteroptera: Miridae). J. Econ. Entomol. 89, 1053-1059 (1996).</li><li>Snoddgrass, G. L., Scott, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discriminating-dose+bioassay+for+detecting+pyrethroid+resistance+in+tarnished+plant+bug+(+Heteroptera:+Miridae)+populations.">A discriminating-dose bioassay for detecting pyrethroid resistance in tarnished plant bug ( Heteroptera: Miridae) populations.</a> Southwest. Entomol. 24, 301-307 (1999).</li><li>Lopez, J. D., Hoffman, W. C., Latheef, M. A., Fritz, B. K., Martin, D. E., Lan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+vial+bioassay+of+insecticidal+toxicity+against+cotton+fleahopper,+Pseudatomoscelis+seriatus+(Hemiptera:+Miridae).">Adult vial bioassay of insecticidal toxicity against cotton fleahopper, Pseudatomoscelis seriatus (Hemiptera: Miridae).</a> J. Pest. Sci.. 33, 261-265 (2008).</li><li>Lopez, J. D., Hoffman, W. C., Latheef, M. A., Fritz, B. K., Martin, D. E., Lan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+toxicity+of+selected+insecticides+against+thrips+on+cotton+in+laboratory+bioassays.">Evaluation of toxicity of selected insecticides against thrips on cotton in laboratory bioassays.</a> J. Cotton Sci. 12, 188-194 (2008).</li><li>Smith, T. P., Hammond, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+susceptibility+of+sweetpotato+weevil+(Coleoptera:+Brentidae)+to+selected+insecticides.">Comparative susceptibility of sweetpotato weevil (Coleoptera: Brentidae) to selected insecticides.</a> J. Econ. Entomol. 99, 2024-2029 (2006).</li><li>Kanga, L. H. B., Wall, M. L., Plapp, F. W., Gardiner, E. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrethroid+resistance+in+field-collected+boll+weevils+from+southeast+Arkansas+in+1994.">Pyrethroid resistance in field-collected boll weevils from southeast Arkansas in 1994.</a> Southwest. Entomol. 20, 247-253 (1995).</li><li>Bayoun, I. M., Plapp, F. W. J. r., Gilstrap, F. E., Michels, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+selected+insecticides+to+Diuraphis+noxia+(Homoptera:+Aphidiae)+and+its+natural+enemies.">Toxicity of selected insecticides to Diuraphis noxia (Homoptera: Aphidiae) and its natural enemies.</a> J. Econ. Entomol. 88, 1177-1185 (1995).</li><li>Smith, T. R., Cave, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+susceptibility+of+Cybocephalus+nipponoicus+and+Rhyzobius+Iophanthae+(Coleoptera+Cybocephalidae,+Coccinellidae).">Pesticide susceptibility of Cybocephalus nipponoicus and Rhyzobius Iophanthae (Coleoptera Cybocephalidae, Coccinellidae).</a> Fla. Entomol. 89, 502-507 (2006).</li><li>Sukontason, K., Olson, J. K., Hartberg, W. K., Duhrkopf, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organophosphate+and+pyrethroid+susceptibilities+of+Culex+salinarius+adults+from+Texas+and+New+Jersey.">Organophosphate and pyrethroid susceptibilities of Culex salinarius adults from Texas and New Jersey.</a> J Am. Mosquito Contr. 14, 477-480 (1998).</li><li>Kanga, L. H. B., Plapp, F. W. J. r., Wall, M. L., Elzen, G. W., Lopez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+for+resistance+to+organophosphorus,+carbamate,+and+pyrethroid+insecticides+in+the+oriental+fruit+moth+(Lepidoptera:+Torticidae).">Monitoring for resistance to organophosphorus, carbamate, and pyrethroid insecticides in the oriental fruit moth (Lepidoptera: Torticidae).</a> Canadian Entomol. , 131-441 (1999).</li><li>Plapp, F. W., Jackman, J. A., Campanhola, C., Frisbie, R. E., Graves, J. B., Luttrell, R. G., Kitten, W. F., Wall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+and+management+of+pyrethroid+resistance+in+the+tobacco+budworm+(Lepidoptera:+Noctuidae)+in+Texas,+Mississippi,+Louisiana,+Arkansas,+and+Oklahoma.">Monitoring and management of pyrethroid resistance in the tobacco budworm (Lepidoptera: Noctuidae) in Texas, Mississippi, Louisiana, Arkansas, and Oklahoma.</a> J. Econ. Entomol. 83, 335-341 (1990).</li><li>Mink, J. S., Boethel, D. J., Leonard, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+permethrin+resistance+in+soybean+looper+(Lepidoptera:+Noctuidae)+adults.">Monitoring permethrin resistance in soybean looper (Lepidoptera: Noctuidae) adults.</a> J. Entomol. Sci. 28, 43-50 (1993).</li><li>Cook, D. R., Leonard, B. R., Gore, J., Temple, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baseline+responses+of+bollworm,+Heliocoverpa+zea+(Boddie),+and+tobacco+budworm,+Heliothis+virescens+(F.),+to+indoxcarb+and+pyridalyl.">Baseline responses of bollworm, Heliocoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), to indoxcarb and pyridalyl.</a> J. Agricul. Urban Entomol. 22, 99-109 (2005).</li><li>Temple, J. H., Pommireddy, P. L., Cook, D. R., Ma&#231;on, P., Leonard, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthopod+management:+Susceptibility+of+selected+lepidopteran+to+pests+Rynaxypyr,+a+novel+insecticide.">Arthopod management: Susceptibility of selected lepidopteran to pests Rynaxypyr, a novel insecticide.</a> J. Cotton Sci. 13, 23-31 (2009).</li><li>Plapp, F. W., Vinson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+toxicities+of+some+insecticides+to+the+tobacco+budworm+and+its+ichneumonid+parasite,+Campoletis+sonorensis.">Comparative toxicities of some insecticides to the tobacco budworm and its ichneumonid parasite, Campoletis sonorensis.</a> Environ. Entomol. 6, 381-384 (1977).</li><li>Xu, J., Shelton, A. M., Cheng, X. i. a. N. i. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+susceptibility+of+Diadegma+insulare+(Hymenoptera:+Ichneumonidae)+to+permethrin.">Variation in susceptibility of Diadegma insulare (Hymenoptera: Ichneumonidae) to permethrin.</a> J. Econ. Entomol. 94, 541-546 (2001).</li><li>Snodgrass, G. L., Scott, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+changes+in+pyrethroid+resistance+in+tarnished+plant+bug+(Heteroptera:+Miridae)+populations+during+a+three+year+period+in+the+delta+area+of+Arkansas,+Louisiana+and+Mississippi.">Seasonal changes in pyrethroid resistance in tarnished plant bug (Heteroptera: Miridae) populations during a three year period in the delta area of Arkansas, Louisiana and Mississippi.</a> J. Econ. Entomol. 93, 441-446 (2000).</li><li>Miller, A. L. E., Way, M. O., Bernhardt, J., Stout, M. J., Tindall, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-state+resistance+monitoring+of+rice+stink+bug+with+a+new+and+old+insecticide.">Multi-state resistance monitoring of rice stink bug with a new and old insecticide.</a> , (2010).</li><li>Abbott, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+computing+the+effectiveness+of+an+insecticide.">A method for computing the effectiveness of an insecticide.</a> J. Econ. Entomol. 18, 265-267 (1925).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> User's Manual version 8.0. , (2002).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A user's guide to Probitor Logit analysis. , (2002).</li><li>Preisler, H. K., Robertson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pesticide bioassays with arthropods. , (1992).</li><li>Hollingsworth, R. G., Steinkraus, D. C., Tugwell, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+Arkansas+population+of+tarnished+plant+bugs+(Heteroptera:+Miridae)+to+insecticides,+and+tolerance+differences+between+nymphs+and+adults.">Responses of Arkansas population of tarnished plant bugs (Heteroptera: Miridae) to insecticides, and tolerance differences between nymphs and adults.</a> J. Econ. Entomol. 90, 21-26 (1997).</li></ol>

LC 50值可用于建立目标人群的易感性（S）为基准。这个数据的价值，可以在今后的监测调查，目前的结果比较以前确定的立法会50确定的目标人群的易感性已转向直接目的。实际LC 50值可以通过检查的95％置信区间种群间的比较;的上限和下限，如果不重叠，那么它很可能人口易感性和在某些情况下经历了显着的变化是一个抵抗的迹象33。立法会50秒 ，也可以用来检查在 28 esusceptibilitye杀虫剂的季节性变化，或比较不同物种或杀虫剂的AI第的回复大量的工作，也可以使用这些数据来比较男性和女性10或成人之间和不成熟的阶段，10,34之间的响应。有时置信区间范围内或无法计算。为了获得更多的昆虫和/或更多的浓度进行更严格的置信区间的生物活性。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>50-100 ml glass beaker </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Volumetric flask with stoppers (can use the amber colored flasks for light sensitive pesticides such as pyrethroids)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>20ml glass scintillation vials with un-lined lids</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A method for color coating vials (paint or markers)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Acetone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Commercial hotdog roller (heat element disconnected)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2 wash bottles (one modified such that the opening is large for fast delivery of the liquid and one so that the spout opening is small for slow delivery of the liquids)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Small weigh boat (make sure all plastic materials acetone safe)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Access to a balance with 0.001g readability or a higher precision.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm M</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Repeater pipettor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Appropriate tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass pipettes that fit in the volumetric flask.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hood (used to remove the acetone smell)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A place to store insecticide solutions and vials [refrigerator (solutions), dark room or freezer depending on the chemical (vials)]</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

bioassays for monitoring insecticide resistance

抗虫性农药是一个日益严重的问题，因为农药生产农业高产的一个不可分割的一部分。当个别害虫标记在一个特定的作物系统的少数产品，化学品控制选项是有限的。因此，同样的产品（S）反复使用和持续的选择压力是对目标害虫。有财政和环境相关的成本与抗性种群的发展。抗药性的费用估计每年约15亿美元在美国。本文将描述协议，目前用于监测节肢动物（特别是昆虫）人口发展的阻力。成人小瓶测试是用来衡量接触杀虫剂的毒性和修改这个测试是植物系统性杀虫剂的使用。在这些生物测定，昆虫接触技术等级杀虫剂和反应（死亡率）在一个特定的暴露后的时间间隔记录。死亡率数据受到登录剂量的概率分析，以生成一个致命的浓度，提供50％的目标人群和一系列的信心限值（CL）的数据变异的估计（LC 50）的死亡率估计。当杀虫剂易感人群范围内收集这些数据，可以用的LC 50为今后的监测目的的基准数据。人口已经暴露的产品后，结果可以比以前确定的LC 50，使用同样的方法。

Watch this Scientific Journal Video about Bioassays for Monitoring Insecticide Resistance at JoVE.com

Bioassays for Monitoring Insecticide Resistance

这个手稿演示和讨论调查农药的易感性和接触电阻和系统性农药检测节肢动物使用的技术。

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 ...

bioassays-for-monitoring-insecticide-resistance

Research

JoVE Journal

Biology

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

测试拟南芥耐咀嚼昆虫的选择和非选择性检测

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

科研

生物学

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

表征草食动物的耐药机制：Spittlebugs臂形 SPP。为例

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

两栖动物卵巢监测和控制的生殖技术

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...