我们感谢 d. 阿什顿, 五. 巴克利、k Beimfohr、f Bojorquez、j 坎特雷尔、g. 卡斯特罗、r. 费恩、Jara、米德、欧文斯、l. Rodarte、d. Sieglaff、Sonoqui、m. Stefanek、b. 斯图尔特、j Trejo、a. 斯莱德和 e. Yescas 技术援助。部分支持是由美国农业部-农业研究局, 美国农业部-国家食品和农业推广 IPM 计划和害虫管理替代方案的特殊项目, 棉花注册, 亚利桑那州棉花种植者协会, 棉花基金会, 美国农业部-人大, NAPIAP (西部地区), 和西部地区 IPM 的特殊项目。

注意: 下面描述的技术被认为是部分生命表, 因为它们没有明确包括成人阶段的复制或死亡。"队列" 一词等同于 "世代", 因为它从卵子到成人阶段的死亡率. 
 1. 建立字段站点 <ol> <li> 一旦出现昆虫, 就在作物生长的任何时候进行生命表。何时开始研究的选择将取决于研究的目标和目标. </li>
	<li> 在绘图中心附近选择两行裁剪, 以最小化周围地块或未开垦区域的边缘效果。用线旗或木桩标记每行的头, 以方便一旦作物变大, 便于搬迁. </li>
	<li> 从与行平行的绘图的边缘移动 3-4 m。使用一行建立卵群, 第二个建立若虫队列 </li> </ol> 2。建立卵群 <ol> <li> 使用8X 手透镜在棉花植株的主茎的顶部 (通常是顶部的第二或第三个节点) 附近的叶子的底部搜索新产卵. 
	注: B. 虱粉 的生命阶段通常垂直分布在植物树冠上, 植物顶部附近有卵, 在下面逐渐若虫阶段。这一结果是因为 1 st 龄若虫与卵在同一叶上定居, 而植物在其生长的初始产卵点上方添加叶子。
	<ol> <li> 使用更高功率的15X 透镜观察新鲜的卵, 并在使用8x 透镜标记昆虫之前验证识别。新鲜的蛋在透镜之下有明亮的白色着色并且从老的蛋站立 ( 图 1A 和 1B )。鸡蛋变暗到 tannish 的颜色, 因为它们成熟. </li>
	</ol> </li> <li> 使用无毒、超细点的黑色永久性标记在鸡蛋周围画一个小圆圈。画出足够小的圆圈以最小化雌性在以后的圆圈中产卵的几率. 
	<ol> <li> 用钢锯或钻头在8X 手透镜侧面切割一个孔或槽, 以便在绘制时可以通过透镜插入和查看钢笔 ( 图 2A ). </li>
	</ol> </li> <li> 如果可能的话, 在同一叶上重复此过程, 以标记其他鸡蛋, 在单个叶子上标记总共不超过四个鸡蛋, 每个叶扇区不超过一个鸡蛋。对于棉花, 叶子被分成四区段由三主要叶子静脉 ( 图 3 ). </li>
	<li> 在包含有标记的蛋的叶子的叶柄周围绑一个小的轻质纸板标签。根据实验设计 ( 图 2B ) 对标记进行编号并包括绘图或处理编号的符号. </li>
	<li> 将1米长的下垂胶带系在靠近植株顶端的主茎附近。使用弓式领带, 使磁带可以很容易地重新定位, 以保持它在现场重复访问该位置可见. </li>
	<li> 在便携式模拟或电子笔记本上记录叶号和位置信息 ( 表 1 )。位置信息使用叶扇区更精细地记录位置 ( 例如 , 1-1、1-2、1-4 表示在扇区1、2和4中的蛋 #1). </li>
	<li> 在一天中建立每个队列. 
	注: 在给定的地块中, 一个队列由至少50个卵总数组成。
	<ol> <li> 每个工厂只使用一张叶子来更好地分配队列中的成员. 
		注: 根据昆虫的密度, 这可能包括任何地方从13叶与3-4 鸡蛋每或50叶与一个鸡蛋每。个别植物被选中, 以尽可能多的行分布标记的叶子. </li>
	</ol> </li> </ol> 3。建立若虫队列 <ol> <li> 使用8X 手透镜搜索新解决的 1 st 幼虫在叶子的底部大约3-5 叶子从棉花植物的主要茎的顶端下来。使用15X 透镜在使用8X 透镜标记之前验证识别. </li>
	<li> 使用无毒、超细的黑色永久性标记在若虫周围画一个小圆圈。画出足够小的圆圈, 以最小化一个爬虫在以后的圈子里定居的机会. 
	注意: 新孵出的 1 st 龄若虫被称为爬行虫, 它可以在卵孵化后的最初几个小时内移动几厘米。然后, #34; 定居和 #34; 到一个网站, 它将饲料和蜕皮, 而不会再次移动, 直到成人出现。这些被安定的 1 st 幼虫若虫 ( 图 1C ) 是独特的从爬行者。首先, 它们是静止的, 其次, 它们是2维的, 在叶子上是扁平的, 并且有稍微半透明的琥珀色。
	<ol> <li> 用钢锯或钻头在8X 手透镜侧面切割一个孔或槽, 以便在绘制时可以通过透镜插入和查看钢笔 ( 图 2A ). </li>
	</ol> </li> <li> 在同一叶上重复此过程, 如有可能, 标记其他若虫, 在单个叶子上标记不超过四若虫, 每个叶扇区不超过一个若虫。对于棉花, 叶子被分成四区段由三主要叶子静脉 ( 图 3 ). </li>
	<li> 在带有标记的若虫的叶子的叶柄周围绑一个小的轻质纸板标签。根据实验设计 ( 图 2B ) 对标记进行编号并包括绘图或处理编号的符号. </li>
	<li> 将1米长的下垂胶带系在靠近植株顶端的主茎附近。使用弓式领带, 使磁带可以很容易地重新定位, 以保持它在现场重复访问该位置可见. </li>
	<li> 在便携式模拟或电子笔记本上记录叶号和位置信息 ( 表 1 )。位置信息使用叶扇区更精细地记录位置 ( 例如 , 1-1、1-2、1-4 表示扇区中的若虫1、2和4在叶 #1). </li>
	<li> 若要确保已结算的 1 st 幼虫若虫和非爬行虫被标记, 请返回每个标记的叶子, 并在初始设置后观察1-2 小时左右的标记昆虫。可能需要对已定居的若虫进行陈述. </li>
	<li> 在一天中建立每个队列. 
	注: 在一个给定的情节中的队列是由至少50若虫总数组成。每株植物只用一片叶子来更好地分配队列中的成员。根据昆虫的密度, 这可能包括任何地方从13叶与3-4 若虫每或50叶与一个若虫每。个别植物被选择分布标记的叶子沿尽可能多的行。由于 1 st 幼虫爬行阶段, 在协议2标记的蛋不是相同昆虫然后被跟随作为若虫因此, 没有测量爬虫的死亡率, 并且生命表在时间上稍有脱节, 因为通常在同一天建立卵和若虫群。研究表明, 爬行的死亡率是微不足道的, 基本上可以忽略 30 . </li>
</ol> 4。卵孵化和死亡的观察和记录 <ol> <li> 在8-10 维 (28-32 和 #176; C; 平均亚利桑那州夏季条件) 在建立卵群后, 收集含有标记的鸡蛋的叶子并返回实验室进行观察解剖显微镜。鸡蛋太小, 无法清楚地评估田间死亡率和死亡率的原因. </li>
	<li> 确定在队列中启动的卵子的死因和记录 ( 表 1 ). 
	注: 死亡的特征是变位、捕食或 inviability。变位: 由于天气事件 (风, 吹尘, 雨) 或咀嚼捕食, 鸡蛋是缺掉的。捕食: 吸吮捕食者留下一个折叠的绒毛膜 ( 图 4K )。孵出的卵可能会出现塌陷, 但卵绒毛膜会有垂直的缝隙。用30-minuten-takt 针在显微镜下的叶子上逗绒毛膜, 寻找这个狭缝。Inviability: 卵在 8-10 d 期后无法孵化, 是深色棕褐色。在亚利桑那夏天情况下 (28-32 和 #176; C) 蛋通常在5-7 天内孵化。这可能在其他区域有所不同, 在收集时间内可能需要从该字段进行调整. </li>
</ol> 5。若虫发育和死亡的观察和记录 <ol> <li> 在队列建立后一到两天, 使用15X 透镜来评估若虫的发育情况, 并在死亡时指定死亡原因。每星期至少做三次观察 (每隔一天). 
	<ol> <li> 使用相对大小 ( 图 1C -g ) 和建立后的时间来评估龄期. 
		注: 有四若虫龄和发展是快速的在亚利桑那夏天情况 (28-32 和 #176; C) 与前三个阶段的每一个持续大约 2 d 和最后阶段持久的 3-5 d (共计若虫发展2星期或更少)。新的观察者应该通过观察在实验室里饲养的昆虫或在寄主植物上的温室来了解幼虫的大小。相对于全身大小而言, bacteriosomes (共生虱) 腹部的相对体积是若虫幼虫的有用指标 ( 图 1C -g )。新蟾的若虫是非常平坦和半透明的。若虫准备蜕皮更肿胀, 圆顶的轮廓, 和不透明的外观. </li>
		<li> 确定若虫死亡原因并记录在队列设置中启动的笔记本 ( 表 1 ). 
		注: 死亡的特点是变位, 寄生, 捕食或未知依赖于幼虫 ( 图 4 )。变位: 任何阶段的若虫由于天气事件 (风、吹尘、雨) 或咀嚼掠夺而失踪。估计被逐出的若虫的阶段作为死亡和活若虫的平均阶段在某一观察日期。寄生: 仅可观察在 4 th 幼虫若虫。配对的淡黄 bacterisomes 由开发的寄生蜂幼虫偏移 ( 图 4A );幼虫阶段有时是可见的 ( 图 4C )。寄生蜂的蛹阶段是独特的和属特定的 ( 图 4B , 4D )。捕食: 吸吮捕食者将疏散若虫的内容, 并留下一个倒下的尸体 ( 图 4G -4I )。很少, 咀嚼捕食者可能留下证据 ( 图 4J )。未知: 死亡不能归因于上述原因之一。在潮湿的环境中, 真菌病可能是另一种死亡原因。这个类别也可能包括由寄生蜂宿主喂养杀死的若虫。幸存的若虫出现在成人留下一个独特的 t 形插槽在人体 ( 图 1H ) </li> </ol> </li> <li> 记录开发阶段 (如果还活着) 和导致死亡和阶段在笔记本中启动的队列建立 ( 表 1 ). </li>
	<li> 一旦所有的若虫在一片叶子上被观察, 要么死亡, 要么成为成年虱, 收集树叶并返回实验室。使用解剖显微镜的更高放大倍数, 确认在现场所注意到的死因是准确的, 并作出任何更正. 
	注: 并非每 non-dislodged 死虫在典型的两周观察期内都将保留在叶子上, 因此无法进行一些验证. </li>
</ol> 6。数据摘要和分析 <ol> <li> 查阅可用的资源以帮助构建生命表从收集的数据 2 、 8 、 11 , 31 。一个示例生命表显示为表 2. 
	注意: 健壮的生命表分析要求在时间和/或不同站点上进行多个独立的生命表。对于像 B. 虱粉 这样的性昆虫, 这可能是一个单一季节和/或多个季节和地点的多个生命表。
	<ol> <li> 估计实际死亡率 ( d x /l 0 ) 根据在世代初建立的昆虫的数量. 
		真正的死亡率 = ( d x /l 0 ) , 其中 d x 是在阶段中死亡的数字 x 和 l 0 是世代开始时的昆虫数量。这些死亡率是累加的, 而 d x 的总和在各个阶段估计了生成的总死亡率 ( 表 2 ). </li>
		<li> 估计在一个阶段 ( q x ) 内的明显死亡率基于在特定阶段开始时活着的昆虫数量 (表 2)。在阶段特定的 q x 中评估特定于阶段的 q x 或因子。这些速率仅在一个阶段内是累加的. </li>
		<li> 使用公式确定边际死亡率: 
		 M B = d b /( 1 维 ) 其中 M b 是死亡率因子的边际率 b, d b 是从因子的明显死亡率 B 和 d 是可战胜因子 B 13 、 32 ( 表 3 ) 的所有死亡因素的明显死亡率总和. 
		注意: 对于像虱和许多其他昆虫一样的无梗昆虫, 在特定生命阶段中死亡的多重原因是不连续的。相反, 他们采取行动同时, 因此, 需要估计边际率, 以准确估计特定阶段的死亡率从任何一个原因 32 , 34 。例如, 寄生蜂可能攻击虱若虫。寄生蜂卵可能孵化, 幼虫可能在寄主中发育。这项活动, 最初无症状的观察员, 做, 或将最有可能杀死宿主昆虫, 应归功于死亡的原因。但在某些情况下, 捕食者可能会攻击同样的若虫, 或者若虫可能会从叶中脱落, 导致观察者注意到死亡的原因是捕食或变位。边际死亡率校正。
		<ol> <li> 将边际阶段特定的速率转换为 k 值 35 为 k =- ln (1- M ), 其中 ln/em>> 是自然对数和 M 是边际死亡率的利息。k 值是加法, 并且这简化进一步分析。k 值可以被转换成比例的死亡率 1- e [-k] 。
		</ol> </li> <li> 估计不可替代的死亡率为 [1- e (-谈谈) ]-[1- e (-{谈谈-kvalue i ) ]。
		注: 如果移除某一特定死亡因素, 这将使总世代死亡率的一部分无法实现。例如, 如果因为杀虫剂喷洒而去除了捕食或寄生, 那么世代死亡率可能会损失多少？无可替代的死亡率估计以这种方式假定没有密度依赖性在死亡率. </li>
		<li> 关键因素 <ol> <li> 使用简单的图形分析来绘制任何一个阶段的 k 值, 或在一个阶段中的任何一个死亡率因子 (或一个死亡率因子) 与整个代的总 k 值 (总计 k = 所有单个 k 值的总和). 
			注: 最接近总 K 型的死亡因素是最重要的因素, 是造成世代死亡率变化最主要的因素 35 。一个更定量的方法退步单个 k 值的总 k, 并确定的关键因素, 作为一个最大的斜率值 3 . </li>
		</ol> </li> <li> 通过对昆虫种群密度的自然对数的兴趣系数进行回归来测试密度依赖性 ( 例如 13 )。一个显著的正斜率表明了直接密度依赖性和负斜率逆依赖. 
		注: 关于成人生存和生殖的附加生命表信息, 许多附加参数 ( 例如 、生成时间、净繁殖率、瞬时增长率、给定阶段的预期寿命、 等 ) 和分析 (矩阵模型和弹性分析 36 , 37 可以进行. </li>
	</ol>
	</li>
</ol>

作者没有什么可透露的。

通常情况下, 性昆虫的生命表的发展被限制在一个垂直的方法, 即人口被反复取样, 然后使用各种图形和数学技术来评估各个阶段的招聘情况, 并根据不同生命阶段的密度变化推断死亡率2。这种方法的优点是, 它通过将一群不动的等龄昆虫从一个种群中隔离出来, 然后随着时间的推移而跟随他们的命运, 来导航这个限制。死亡率可以直接估计, 同样重要的是, 可以确定这种死亡率的代...

生命表是一个在生态学中具有悠久历史的常用工具1,2。生命表实质上是一个时间内的人口出生率和死亡率的时间表, 这些数据可以用来量化一些重要的参数来理解和预测人口动态。生命表还可能提供有关死亡原因的信息, 这些因素对于了解营养相互作用以及制定农业和自然系统中害虫管理的控制策略都很重要。已经为昆虫搭建了许多实地的生命表3,4,5, 分析为许多管理和预测昆虫种群的动态、调控和预报提供了重要的洞察力。自然系统6,7,8,9,10,11,12,13,14."生命表" 一词也经常用于描述基于实验室的研究, 主要是检查出生和死亡的时间表, 但在人工条件下, 不会使昆虫暴露在自然死亡的力量和现实环境变量中。一般来说, 实验室研究的目的是估计一个物种的相对生物潜能。这里描述的方法的重点是针对基于字段的调查, 它定义了与环境相关的实现潜力。
生命表可以被描述为水平, 在这种情况下, 一个真正的平等年龄的人从他们的生命开始, 直到死亡, 或垂直, 经常样本是通过时间的人口与假设稳定的年龄结构然后从数学构造的队列中推断出生命率2,15。可以部署的生命表的类型取决于昆虫的性质。水平寿命表通常可以开发为 univoltine (每年一代) 昆虫, 而这种方法可能是非常具有挑战性的性昆虫与多和广泛重叠的世代每年。已经提出了许多分析方法, 并用于开发昆虫种群的垂直生命表 (参见伍德2 )。这里展示的方法允许开发 cohort-based, 水平的生活表在领域为性昆虫以具体生活历史特征, 特别是, 存在无梗生命阶段。对棉花主要害虫的模型体系进行了研究。
虱, 烟粉虱型 B (= 烟 argentifolii, 中东-亚洲小 116) 是一种全球农业害虫, 对许多农艺和园艺作物的产量和质量产生负面影响, 包括温带地区的农业保护系统17。影响发生的原因是韧皮部喂养扰乱了养分流动, 若虫喂养造成的不明病因失调, 许多植物病毒的传播和作物质量的影响由于甘露的沉积18,19.昆虫有一个宽广的寄主范围并且是性的, 有多达12-13 世代每年根据区域和可利用的食物资源20。管理方面的挑战也因其高的生殖潜力、其在农业系统内和之间的分散和迁移能力、缺乏静止阶段 (滞育或夏眠) 及其对迅速发展抗药性的处置而加剧。用于用于抑制21,22的杀虫剂。
在制定病虫害综合防治战略以有效和经济地管理受影响作物的害虫种群方面取得了相当大的进展23,24,25。这些管理系统是基于对B. 虱粉的种群动态的基本理解, 而生命表是使这一理解得以实现的关键技术。在亚利桑那州, 生命表允许在多个作物系统中对B. 虱粉的重要死亡率进行估计和识别1326, 使死亡率动态的测量相对于管理策略, 包括杀虫剂的非靶标效应14, 提供了一种估计潜在的功能非靶标的方法转基因棉生产杀虫蛋白的效果27, 支持严格评估一个经典的生物控制程序28 (纳兰霍, 未发布的数据), 并帮助探索自上而下和自下而上的效果对害虫动态的比较效果29。所有这些应用程序都部署了此处描述的方法。该方法可用于研究一些自然和管理系统中的昆虫种群生态学。

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methodology for developing life tables for sessile insects in the field using the whitefly, bemisia tabaci, in cotton as a model system

生命表提供了一种方法来测量随着时间的推移人口出生和死亡的时间表。它们还可用于量化人口死亡率的来源和比率, 在生态学中有各种应用, 包括农业生态系统。水平, 或 cohort-based, 生命表提供了最直接和准确的量化关键人口率的方法, 因为他们跟随一群人从出生到死亡的人口。在这里, 我们提出的协议, 以进行和分析 cohort-based 生命表的领域, 利用无梗性质的不成熟生命阶段的全球害虫, 烟粉虱。个体昆虫位于棉叶的底面上, 通过用无毒的钢笔在昆虫周围画一个小圆圈来标记。随着时间的推移, 这种昆虫可以被反复观察, 用手部镜片来测量从一个阶段到下一个时期的发展, 并确定与自然和引入的死亡力量相关的特定阶段死亡原因。分析解释了如何正确地测量在每个阶段同时的多重死亡力量, 以及如何使用这些数据来提供有意义的人口动态指标。该方法不直接考虑成人的生存和繁殖, 从而限制了对未成熟阶段动力学的推断。一个例子是, 侧重于测量的影响, 自下而上 (植物质量) 和自上而下 (天敌) 影响的死亡率动态的B. 虱粉在棉花系统。

表 2中提供了一个队列示例, 以显示生命表结果的典型表示和计算。最有用的数据是在每个阶段的每个因素的边际死亡率中捕获的。通过将这些比率换算成 k 值 (议定书6节), 所有各阶段的特定阶段死亡率和所有因素的具体死亡率都可以很容易地估计出来, 就像总世代死亡率一样。这也促进了不可替代的死亡率、关键因素和密度依赖性分析。
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Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

生命表允许定量的来源和死亡率的昆虫种群, 有助于理解, 预测和操纵人口动态在生态。本文介绍了在无梗幼生物期的昆虫田间 cohort-based 生命表的传导和分析方法。

用虱、烟粉虱在棉花中开发无梗昆虫生命表的方法作为模型系统

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

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Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

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11.8K 次观看.  USDA-ARS, Arid-Land Agricultural Research Center. 生命表允许定量的来源和死亡率的昆虫种群, 有助于理解, 预测和操纵人口动态在生态。本文介绍了在无梗幼生物期的昆虫田间 cohort-based 生命表的传导和分析方法。

视频: Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

一个反对臭虫评估杀虫剂效果的方法，<em&gt;臭虫臭虫</em&gt;，鸡蛋和一龄期

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

科研

环境科学

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

遗传学

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

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

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

协议侦办主机组织分布，传播模式和效果上浓核病毒的主机健身的棉铃虫

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

免疫与感染

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13, ecology2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14 or compounds15, new cultural approaches1,4,19, or the selection and development of resistant cultivars7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.
Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.
There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.

Improving our understanding of and our ability to manage many of the insect-transmitted plant viruses requires the use of the vector. Insect transmission of plant viruses is a tritrophic interaction, and as such requires the manipulation of insects, virus, and plant. Vectors must be reared in large numbers and manipulated in such a way as to insure high rates of transmission to test plants. The basics of rearing and manipulating the whitefly, Bemisa tabaci (Hemiptera: Aleyrodidae), a vector of plant viruses belonging to several emerging plant virus genera, representing a large number of economically significant viruses is presented.

发射使用粉虱的植物病毒

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

免疫荧光和定量PCR对白蝇组织中白蝇病毒的定位和定量

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

从白蛉提取内分泌物的方法<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

局部应用生物测定以量化蚊子和果蝇的杀虫剂毒性

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

通过饲喂测定评估植物化学对 Helicoverpa armigera 的影响

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

作者聚焦:害虫管理饲喂分析的发展和挑战 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

开发一种用于评估植物化学物质对 Helicoverpa armigera 的杀虫作用的摄食试验系统

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

用虱、烟粉虱在棉花中开发无梗昆虫生命表的方法作为模型系统

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一个反对臭虫评估杀虫剂效果的方法，