我们感谢艾拉 Weinert, 米歇尔赫尔姆斯和 Marlis Badusche 为优秀的技术援助, 杰西卡 Börstler 分析淡色蚊子, 罗伯特贝克尔和 Björn Pluskota 提供白纹伊蚊蛋和为 Vapalahti提供病毒储存。ML 得到了莱布尼茨协会的支持;授予编号 SAW-2014-SGN-3。

1. 准备雌性蚊子
<ol><li>使用一个吸引器 (大约400蚊子每大笼子) 收集蚊子从一个大笼子。</li>
	<li>麻醉蚊子与二氧化碳 (CO2) 为 7 s。</li>
	<li>每塑料瓶20位女性 (Ø50毫米 x 100 毫米)。 注: 如果蚊子醒了, 使用另外 3 s CO2.
</li>
	<li>一夜之间饿死蚊子。</li>
</ol>2. 人工血粉
注意: 所有步骤都在 BSL-3 条件下执行。
<ol><li>
准备传染性的血餐。
	<ol>
<li>将病毒储存稀释至 1 x 108斑块成型单元 (PFU)/毫升浓度。 注意: 通过执行组织培养感染剂量50方法 (TCID50), 并由长矛 & Kärber 算法10,11计算出库存浓度。</li>
		<li>混合过期的人血 (血液保存) 从血库 (不适合人类, 但有用的蚊子) 与果糖 (8% 溶液), 过滤牛血清 (血清) 和工作溶液的病毒储存 (过期的人类 blood:fructose:FBS:工作解决方案 = 5:3: 1:1)。 注: 我们选择了人类的血液, 因为这是已知的天然哺乳动物宿主的 ZIKV。</li>
	</ol>
</li>
	<li>冷冻140µL 的血粉混合供进一步分析通过 TCID50。</li>
	<li>
使用不同的喂养方法进行人工喂养, 具体取决于种类:
	<ol>
<li>
白纹蚊: 使用膜喂养系统30分钟 (每馈线1毫升)。</li>
		<li>
淡色: 一夜之间通过棉花棒提供传染性血液, 不加热血液 (每瓶300µL)。</li>
		<li>
白纹伊蚊: 把两滴 (50 µL) 的血餐混合到塑料瓶中, 让蚊子喂养2小时。不要加热血液。 注意: 蚊子会在头15分钟内进食。由于安全原因, 长时间的潜伏期是必要的。血滴在2小时后排出, 因此在喂食蚊子的分类过程中减少了污染风险。</li>
	</ol>
</li>
	<li>麻醉蚊子与 CO2为 7 s。 注: 如果蚊子醒了, 使用另外 3 s CO2。</li>
	<li>排序和计数完全肿胀蚊子进入一个新的瓶子。</li>
	<li>添加一个棉垫, 饱和与果糖 (8% 溶液) 之间的瓶子和插头。</li>
	<li>保持蚊子在指定的温度18°c 或27°c 在湿气80% 为14或21天。</li>
	<li>用果糖饱和的棉垫喂蚊子。每瓶72小时补充1.5 毫升果糖 (8% 溶液) (最多20只蚊子, 取决于喂养速率, 见步骤 2.5)。</li>
</ol>3. 14 或21天强迫流涎化验
注意: 所有步骤都在 BSL-3 条件下执行。
<ol><li>种子 2 x 104维罗细胞/每井在96个井板与200µL 生长培养基 (Dulbecco´s 修饰的老鹰培养基 (DMEM) 补充3% 血清, 100 U/毫升青霉素, 100 毫克/毫升链霉素, 5 µg/毫升两性霉素 B, 2 毫米 l-谷氨酰胺, 1% 非必需氨基酸 (NEAA), 1% 丙酮酸钠)。</li>
	<li>
准备流涎设备:
	<ol>
<li>将方形玻璃板 (20 x 20 厘米) 放置在工作台上的一个角度。 注意: 这是必要的, 因为冲击波在安全实验室。如果板材是水平或垂直的, 液体将泄漏。</li>
		<li>将双面胶带放在玻璃板的顶部。</li>
		<li>作用的建模粘土, 直到它达到直径0.5 厘米, 并将其水平1厘米, 远离双面胶带。</li>
		<li>切断10µL 过滤尖端尖端的前0.3 厘米。 注: 建议在生物安全实验室以外的地方开始实验前做好准备。</li>
		<li>用10µL 磷酸盐缓冲盐水 (PBS, pH 值 7.4) 填充过滤器提示。</li>
		<li>将过滤器提示放在建模粘土上, 并向胶带倾斜。</li>
		<li>使用柔和的压力来保护过滤器提示。</li>
	</ol>
</li>
	<li>
准备蚊子:
	<ol>
<li>麻醉蚊子与 CO2。</li>
		<li>去掉腿和翅膀, 使蚊子不动。</li>
		<li>把活的蚊子尸体固定在过滤器尖端上面的粘胶带上。</li>
		<li>将喙轻轻放进过滤器尖端。</li>
		<li>运行流涎化验30分钟。</li>
		<li>删除过滤器提示, 丢弃粘土和胶带。</li>
	</ol>
</li>
</ol>4. 唾液的处理
注意: 在 BSL-3 条件下执行 4.1–4.6 2 步。
<ol><li>将这些内容排出到含有10µL 的反应管中。</li>
	<li>轻轻混合, 并将内容转移到准备好的96井板的水井中, 每样都用一个井。</li>
	<li>孵化板材7天在37°c 与 5% CO2。</li>
	<li>使用双目检查细胞是否存在病变效应 (CPE)。</li>
	<li>如果该井是积极的 CPE, 收获上清的吹打140µL 成一个反应管的 RNA 提取。</li>
	<li>
使用 rna 提取试剂盒纯化 rna。
	<ol>
<li>混合140µL 的上清与560µL 的 AVL 缓冲 (病毒裂解缓冲) 和孵化10分钟室温。</li>
		<li>添加560µL 乙醇 (96%) 和涡流样品。 注: 此步骤后, 样品被灭活, 可以出院的 BSL-3 实验室。</li>
		<li>吸管630µL 的样品到一列和离心机与 6000 x g 为1分钟。</li>
		<li>丢弃滤液, 吸管剩余的630µL 在柱上, 重复离心。</li>
		<li>将500µL 的洗涤缓冲器1加到柱上, 离心机在 6000 x g处进行1分钟, 将 RNA 与柱膜结合。</li>
		<li>丢弃收集管并将该列放入新的收集管中。添加500µL 洗涤缓冲2和离心机在 12500 x g为3分钟。</li>
		<li>丢弃收集管并将该列放入1.5 毫升反应管中。</li>
		<li>添加50µL 洗脱缓冲液, 在室温下孵育1分钟。</li>
		<li>离心机在 6000 x g 1 分钟, 然后丢弃该列。</li>
	</ol>
</li>
	<li>通过 RT-qPCR 分析 ZIKV RNA 的样品。</li>
</ol>5. 蚊子身体的加工
注意: 步骤5.1–5.5 是在 BSL-3 条件下执行的。
<ol><li>除去蚊子的身体, 把每个身体放到一个反应管里。 注意: 此时可以暂停实验, 并将样品存储在摄氏-80 摄氏度。</li>
	<li>在反应管中加入500µL 均匀介质 (DMEM 无任何 supplementals)。</li>
	<li>融汇使用手马达搅拌机, 并为每个样品使用新的雌蕊。</li>
	<li>将匀浆的200µL 加入96井板进行提纯。 注意: 此时可以暂停实验, 并将样品存储在摄氏-80 摄氏度。</li>
	<li>在60摄氏度处孵化60分钟, 使板材失效。</li>
	<li>通过全自动核酸提纯纯化 RNA。 注意: 此时可以暂停实验, 并将样品存储在摄氏-80 摄氏度。</li>
</ol>6. 分析
<ol><li>使用 RT qPCR 量化病毒 RNA 拷贝。 注意: RNA 拷贝是平均的在所有 ZIKV 正面蚊子身体和不包括 ZIKV 阴性蚊子。</li>
	<li>计算 IR, 定义为 ZIKV 阳性蚊子身体的数量每位喂养的女性。</li>
	<li>计算 TR, 定义为每数量 ZIKV 阳性的蚊子身体 ZIKV 阳性唾液的数量。没有 ZIKV 阳性体的物种温度组合不能计算出传输速率。</li>
</ol>

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作者没有什么可透露的。

以前已经表明, 强迫流涎获得的结果与经典的再喂养实验6一致。然而, 无论是在重新喂养实验和我们提出的强迫流涎的方法, 它是不可能直接证明流涎活动或监测唾液释放。为了证明流涎的活性, 样品可以检测唾液中的其他成分 (例如,蛋白质, 碳水化合物等)。此外, 额外的 qPCRs 将允许检测病毒 RNA 拷贝。然而, 这需要在不同的分析中分离唾液样本, 这可能会限制细胞培养实验中极低颗粒数的敏感性。反过来, 这可能导致低估了已确定的传输速率。
为了重现的结果, 有必要确保所有蚊子活着直到实验结束。这是通过视觉监测蚊子的身体运动活动来控制的。此外, 对某种蚊子物种的流涎试验的原则使用必须通过喂养控制病毒进行测试, 已知这类蚊子是由这种特定的蚊虫传播的。反之亦然, 在实验中引入的每种病毒都必须在易感蚊子中进行测试。
强迫流涎测定有许多优点。高数量的蚊子可以同时测试在一个单一的标本基地在控制标准化的条件下, 而不与动物福利条例9的冲突。
该方法由几个实验室使用。然而, 确切的设置可能会有所不同, 这可能导致实验室之间的结果差异。一个关键的组成部分是毛细管收集唾液, 可以由玻璃14或塑料15。为了符合安全等级 3 insectary 的高安全性规定, 我们使用了塑料过滤器的小贴士代替了玻璃毛细血管, 从而降低了受伤的风险。此外, 过滤器提示有优势, 收集的液体可以很容易地转移使用吸管。
这里提出的强制流涎试验的设置是基于两个调查人员一起工作和分担职责。蚊子的复员是由一个人执行的, 另一个则同时准备强制流涎设置。这大大减少了处理时间, 并减少了样本切换的风险, 因为在复员工作场所和流涎之间的变化是不必要的。此外, 它允许在复员后立即将蚊子放在强制流涎装置上。这相当短的蚊子处理时间是必不可少的成功强迫流涎。最后, 在整个实验中可以直接监测蚊子的运动。
本文介绍的流涎分析方法是为了深入了解蚊虫的载体电位。然而, 为了回答其他一些具体问题 (例如, 感染脊椎动物所需的蚊子叮咬数量), 动物实验可能仍然需要。

在 2015年, Zika 病毒 (ZIKV) 是属于家庭Flaviviridae的一种蚊媒病毒 (mobovirus), 出现在哥伦比亚和巴西, 在美洲和加勒比地区迅速蔓延, 引起了大量相关的流行病。小头和 Guillain–Barré综合征1例临床病例。蚊和白纹伊蚊的蚊子被认为是 ZIKV2的主要和次要载体, 但白纹伊属的其他种类也被证明具有实验向量能力3.与伊蚊不同的是, 迄今为止测试的大多数淡色物种都无法传播病毒4。只是,淡色致倦的数据提供了不确定的结果3。目前, 在中等温度条件下 (< 20 摄氏度), ZIKV 的蚊媒能力缺乏信息。然而, 这一信息对于可能蔓延到诸如中欧等温带气候地区的风险评估是很重要的。
ZIKV 的有效载体能够获得、维持、放大并最终传播病毒。因此, 对 ZIKV 的蚊体或身体部位, 包括腿部、中肠、头或涎腺进行检测, 不足以确定载体的能力。在释放的唾液中检测感染病毒微粒是强制性的。为了评估向量的能力, 两种感染率 (IR, 每数量的 ZIKV 阳性蚊子的数量) 和传播率 (TR, 蚊子数量与 ZIKV 阳性唾液每数量 ZIKV 阳性蚊子身体), 必须确定。蚊子的 IRs 可以很容易地通过简单的均质蚊子分析, 其次是反向逆转录酶链反应 (RT qPCR) 靶向 ZIKV。此外, 感染还可以通过确定每只蚊子的病毒拷贝数来进一步表征。相比之下, 传输速率的分析依赖于在个体蚊子唾液中检测出可行的病毒微粒。这可以通过喂养受感染的蚊子在敏感的寄主动物, 其次是分析的病毒血症在主机5。然而, 这种方法依赖于合适的模型有机体, 而且代价高昂, 受到动物福利法规的限制。为了避免使用实验动物来分析传输速率和降低成本, 使用血滴的人工再喂养系统被描述了6。然而, 个体规模的测试与大量个体的结合是困难和耗时的。流涎的第一个描述在个体尺度被出版了在 1966年7年, 并且在近几年, 强迫流涎实验成为了评估蚊子的媒介能力的选择方法3,8.
根据我们的媒介能力研究最近出版的中欧蚊子9, 我们描述强迫流涎报告由安德森et。8与一些修改。该方法允许高生物安全水平条件下的高通量标准化实验 (BSL-3), 包括基于细胞培养的活性病毒的鉴定。这里所描述的实验包括856只蚊子。他们通过人工血餐感染 ZIKV, 随后对唾液中感染性病毒微粒的存在进行分析。在实验中引入的蚊子包括两个长期建立的 Ae 的实验室种群. 蚊和淡色库蚊虱molestus (Cx molestus), 以及田间捕捉淡色库蚊虱库蚊 (cx),淡色 torrentium (cx torrentium) 和两个Ae. 伊蚊种群。除了其中的两个Ae.在意大利收集的伊蚊种群中, 所有蚊子都是在德国收集的。

<table><tbody><tr><td>Inject+matic Sleeper</td><td>Inject+matic</td><td></td><td></td></tr><tr><td>Bugdorm-1 Insect Rearing Cage</td><td>BugDorm Store</td><td></td><td></td></tr><tr><td>Drosophila cultivation tubes</td><td>carl roth</td><td>PK11.2</td><td>plastic vial for mosquitoes</td></tr><tr><td>Plug for drosophila cultivation tubes</td><td>carl roth</td><td>PK15.1</td><td></td></tr><tr><td>Constant climate chamber</td><td>Binder</td><td>KBF-240</td><td></td></tr><tr><td>Blood</td><td></td><td></td><td>banked human blood, expired, not usable for medical application</td></tr><tr><td>Dumont-Pincette Electronic SS</td><td>Dumont</td><td>B40c</td><td></td></tr><tr><td>Vero cells</td><td>BNITM</td><td></td><td></td></tr><tr><td>Flash light aspirator</td><td>Bioquip</td><td>2809</td><td></td></tr><tr><td>double-sided adhesive tape</td><td></td><td></td><td>no specific provider</td></tr><tr><td>Gammex Powder-free gloves with AMT</td><td>Ansell</td><td></td><td>for safety reasons necessary during work with pricks in the BSL3 laboratory</td></tr><tr><td>RealStar Zika Virus RT-PCR-Kit</td><td>altona diagnostics</td><td>591013</td><td></td></tr><tr><td>MagMax Pathogen RNA/DNA Kit</td><td>Thermo fisher scientific</td><td>4462359</td><td>RNA isolation of mosquito bodies</td></tr><tr><td>Qiamp viral RNA Mini Kit</td><td>Qiagen</td><td>52906</td><td>RNA-Isolation of supernatant</td></tr><tr><td>filter tips (20 &amp;micro;L)</td><td>Sarstedt</td><td>701,116,210</td><td>Cut the first 3 mm of the tip</td></tr><tr><td>hand motor mixer</td><td>carl roth</td><td>sold out</td><td></td></tr><tr><td>micro pestles for hand motor mixer</td><td>carl roth</td><td>CXH8.1</td><td></td></tr><tr><td>DMEM</td><td></td><td>P0403550</td><td></td></tr><tr><td>L Glutamine Penicilin/Streptomycin</td><td>PAN</td><td>P0819100</td><td></td></tr><tr><td>Amphotericin B</td><td>PAN</td><td>P06-0110</td><td></td></tr><tr><td>Fructose</td><td>carl roth</td><td>4981.5</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>PAN</td><td>P04-36500</td><td></td></tr><tr><td>FBS</td><td>PAN</td><td></td><td></td></tr><tr><td>Sodium Pyruvat</td><td>PAN</td><td>P0443100</td><td></td></tr><tr><td>MEM NAA</td><td>PAN</td><td>P0832100</td><td></td></tr><tr><td>Hemotek Feeder</td><td>Hemotek</td><td>PS6</td><td></td></tr><tr><td>Light Cycler</td><td>Roche</td><td>480 II</td><td></td></tr><tr><td>Light Cycler Software</td><td>Roche</td><td>480 SW 1.5.1</td><td></td></tr><tr><td>Modeling clay</td><td></td><td></td><td>no specific provider</td></tr><tr><td>King Fisher Flex Purification System</td><td>ThermoFisher scientific</td><td></td><td></td></tr><tr><td>&lt;em&gt;Aedes albopictus&lt;/em&gt;</td><td></td><td></td><td>eggs were collected in Freiburg, Germany</td></tr><tr><td>Binocular</td><td>MOTIC</td><td>SMZ-168</td><td></td></tr><tr><td>Binocular light</td><td>MOTIC</td><td>MLC-150C</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-Incubator</td><td>Thermo scientific</td><td>MIDI 40</td><td></td></tr></tbody></table>

forced salivation as a method to analyze vector competence of mosquitoes

向量能力被定义为蚊子种类的潜力传播蚊子传播的病毒 (mobovirus) 到脊椎动物宿主。有效的病毒粒子通过感染蚊子的唾液在血液中传播。强迫流涎化验允许在单蚊子的基础上确定矢量电位, 避免使用动物实验。该方法适用于在短时间内对大量蚊虫进行一次实验分析。采用强迫流涎测定法分析了德国境内856只蚊子, 包括两种不同的淡色库蚊虱、淡色 torrentium和白纹伊蚊,实验感染与 Zika 病毒 (ZIKV) 和孵化在18°c 或27°c 为两个和三周。结果表明, 不同淡色类群对 ZIKV 的载体能力缺乏。相比之下,白纹伊蚊易受 ZIKV 的影响, 但只有27摄氏度, 传播速率类似于白纹伊蚊蚊实验室菌落平行测试。

白纹蚊被称为 ZIKV 的有效载体, 至少在热带气候条件下为12。因此, 我们使用Ae. 蚊作为一个积极的控制, 以建立检测和分析 IRs 以及在孵化温度为27°c 和湿度为80%。IR 和 TR 被定义为以前描述的财神等。13. 挑战Ae. 蚊显示了50% 和72% 的 IR 和42% 和31% 在14和21天后感染 (dpi), 分别 (图 1A和1B)。随后, 欧洲蚊子被测试 ZIKV 感染和传播使用热带和温带气候条件。
在27摄氏度的孵化温度下, 所有蚊子种类显示 ZIKV 感染在 14 dpi, 以及 21 dpi, 除了cx. 库蚊和cx. torrentium的个人, 这是积极的 14 dpi 仅。与不同的淡色类群相比, 所有的伊蚊物种都显示出较高的 irs (介于50% 和72% 之间) 以及较高的病毒 RNA 浓度 (106至 109 RNA 拷贝/蚊子标本), 这表明 irs 范围介于0% 和32% 和病毒装载 103到 104 RNA 拷贝或蚊子标本。所有的伊蚊物种都显示 ZIKV 阳性唾液 (TR 13–42%), 而淡色类群中的唾液样本没有 ZIKV (图 1A和1B)。有趣的是, 21 dpi 的传输速率在 ae 中是相似的. 蚊和ae从德国 (30%), 但极大地低在ae.来自意大利的伊蚊 (13%)。
在18摄氏度的适度温度下孵化有挑战的蚊子, 在白纹病毒的种类 (104–106 RNA 拷贝/蚊子) 以及在不同的淡色分类群 (102–104RNA 拷贝或蚊子)。然而, 在18摄氏度的孵化温度下, 没有观察到任何被调查的蚊子的 ZIKV 的传播。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57980/57980fig1.jpg"> 图 1: 实验性感染 ZIKV 的蚊子的传播速率.ZIKV 在不同蚊虫种类中的载体能力研究结果。六种不同的蚊子分类感染了 ZIKV, 在27摄氏度或18摄氏度处孵化 14 (A) 或 21 (B) 天。TR 被定义为蚊子的数量与 ZIKV 阳性唾液每数量 ZIKV 阳性蚊子身体。调查的蚊子总数为856人, 每个时间点/温度/蚊子分类的人数最少30。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57980/57980fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes at JoVE.com

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

强迫流涎法分析蚊虫的载体能力

为有效控制蚊媒传播病毒, 有关蚊子的矢量电位的知识尤其值得关注。本文将强迫流涎作为一种分析白纹伊蚊和三种不同淡色类群对 Zika 病毒传播的载体能力的方法。

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

forced-salivation-as-method-to-analyze-vector-competence

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.5K 次观看.  Bernhard Nocht Institute for Tropical Medicine. 为有效控制蚊媒传播病毒, 有关蚊子的矢量电位的知识尤其值得关注。本文将强迫流涎作为一种分析白纹伊蚊和三种不同淡色类群对 Zika 病毒传播的载体能力的方法。

视频: Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

甲病毒转导系统：可视化，以免蚊虫感染的工具

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

科研

免疫与感染

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

环境科学

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

发育生物学

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

接种<em&gt;冈比亚按蚊</em&gt;珠蚊子诱发和测量黑化免疫反应

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

经口喂养和显微注射对成年蚊子进行实验性病毒感染

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

生物学

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. ...

Topical Testing Assay: A Technique to Evaluate the Insecticidal Potential of Dopamine Receptor Antagonists on Adult Female Mosquitoes by Topical Application

Culture 3 to 5-day-old adult female mosquitoes in a 20-liter plastic cage. Label 9-ounce paper cups with the name and concentration of testing compound.
Next, prepare a 10 &#181;g/&#181;L stock solution of the testing compound in acetone in a 20-milliliter glass vial. Then, serially dilute the stock solution to obtain the desired working concentrations.
Clean a 1-milliliter glass syringe with acetone, and then, fill it with the test solution at the appropriate concentration. Secure the syringe in a micro-applicator, adjusted to deliver a volume of 0.25 microliters.
Next, use an aspirator to remove ten 3 to 5-day-old adult female mosquitoes from the cage, and anesthetize them for 5 minutes at 4 degrees Celsius. Then, transfer the mosquitoes to a Petri dish, and place the dish on ice for 10 minutes.
In order to avoid damaging the adult mosquito, which could contribute to mortality, ensure that they are not anesthetized in the fridge for more than 5 minutes, or immobilized on ice for more than 10, and minimize the handling of the mosquitoes. If you have access to a cold plate and a micromanipulator with a movable stage, that may further minimize handling of the mosquitoes.
Use fine tweezers to remove each mosquito from the dish. With the syringe micro-applicator, apply 0.25 microliters of testing solution to the dorsal thorax under a dissecting microscope. Next, transfer the mosquitoes to a labeled paper cup on ice. Seal the cup with a 10 by 10-centimeter mesh square, and a rubber band. Then, transfer it to the growth chamber, and record the number of dead mosquitoes.

强迫流涎法分析蚊虫的载体能力

摘要

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此视频中的章节

甲病毒转导系统：可视化，以免蚊虫感染的工具

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

接种

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

使用寨卡病毒对埃及伊蚊的载体能力分析

经口喂养和显微注射对成年蚊子进行实验性病毒感染

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药

局部应用生物测定以量化蚊子和果蝇的杀虫剂毒性

局部测试试验:一种通过局部应用评估多巴胺受体拮抗剂对成年雌性蚊子的杀虫潜力的技术

强迫流涎法分析蚊虫的载体能力

摘要

探索更多视频

此视频中的章节

甲病毒转导系统：可视化，以免蚊虫感染的工具

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

接种

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

使用寨卡病毒对 埃及伊 蚊的载体能力分析

经口喂养和显微注射对成年蚊子进行实验性病毒感染

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

局部应用生物测定以量化蚊子和果蝇的杀虫剂毒性

局部测试试验:一种通过局部应用评估多巴胺受体拮抗剂对成年雌性蚊子的杀虫潜力的技术

使用寨卡病毒对埃及伊蚊的载体能力分析

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药