Name: physiological recordings and rna sequencing of the gustatory appendages of the yellow-fever mosquito aedes aegypti
Uploaded: 2014-12-30T14:00:00.000+00:00
Duration: 9 min 9 s
Description: Watch this Scientific Journal Video about Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti at JoVE.com

The authors thank Bryan T. Vinyard of the USDA, Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Research Center, Biometrical Consulting Service, Beltsville, MD for statistical analyses. This work was supported in part by a grant to J.C.D. from the Deployed War Fighter Protection (DWFP) Research Program funded by the Department of Defense through the Armed Forces Pest Management Board (AFPMB).

1.饲养阂。伊蚊成人<ol><li>孵化的鸡蛋在浅盘约¾英寸的水。拥挤会降低成人的尺寸。 </li><li>后排幼虫在25°C（12-HL:12-HD）和饲料与地面的鱼食。 注:过度喂食可能会降低存活率。 </li><li>每天单独删除蛹由巴斯德吸管，并转移到塑料菜细网状盖（9厘米×5.5厘米）内的小水桶遏制，从而确立了24小时的年龄组。 </li><li>由棉花球进料成蚊10％蔗糖溶液置于笼壳体的屏壁上下同一光周期作为幼虫的蚊子在环境室中于27℃和70％相对湿度。 </li><li>对于电，用5-10日龄的动物。在一般情况下，较大的动物将产生更好的结果。对于RNA分离，可使用动物内，在5-10天龄的范围内的单个24小时年龄分组那个。 </li></ol><ol><li>确定电解质（ 如 1-10毫米）的引出最小的活动，从选定的感觉神经元的适宜浓度。 10毫米氯化钠或氯化钾为1mM是合理的电解质时，开始了一个实验来测试基线活动。 </li><li>溶解各实验化学在合适的溶剂（如果不是水，使用乙醇或DMSO）相比单独电解质对尖峰活性几乎没有或没有影响。最终浓度10％的乙醇或1％DMSO中是合理的起始浓度。 </li><li> （苦或蔗糖甜如奎宁）昆虫是很好的描述喂养威慑或兴奋剂控制化学品选择合适的。 </li></ol> 3.电生理学（尖记录8;图1） <ol><li>表玻璃电极通过机械拉玻璃毛细管。优化的热量和拉设置拉玻璃形成适当形状的recordi吴电极。小心使用以创建电极尖端开口是足够宽，以包络目标感器，但是足够小，以避免与邻近结构接触在解剖显微镜下用镊子折断玻璃电极的末端。 </li><li>在显微镜下，用视觉形态识别和定位目标感器/感器，以确保记录头可重复性。准备动物允许从电极进入角度自由进入感器。 </li><li>冷麻醉成虫在冷冻在-20℃。避免外周神经元损害过度暴露于寒冷的气温。切透明胶带窄条与载玻片刀片。固定使用狭长载玻片全虫。 </li><li>将化学削尖的钨丝放入背胸部或眼睛充当冷漠的（地）电极。 </li><li>填在步骤3.1与感兴趣刺激溶液玻璃电极。使用插入注射器，瓶盖填写从拉到底illary钝结束。轻弹所有气泡，拿着拉降到底。插入一个银线（氯化可选）到在步骤3.1使玻璃电极作为记录和刺激电极。 </li><li>连接电极是专为录音从昆虫接触化学感受性感器前置放大器。此前置放大器（300 Hz至3 kHz的带通滤波器）将减少沉降时间捕捉到神经元的活动接近刺激引进越好。收集，存储和分析使用装备有穗记录软件的微计算机的电信号。 注意:正确接地的记录设置可以大大提高信噪比。 </li><li>刺激感受器与控制解决方案（仅电解质），以确保功能。数起200毫秒后的刺激伪迹的所有准备工作尖峰。 </li><li>随机化的测试化学品的顺序对所有的实验中，除了剂量响应研究中，消除偏见。对于剂量响应研究中，暴露感器增加了实验化学浓度。允许一个最小的刺激之间3分钟，以确保有足够的恢复。 注:此要​​求可能因昆虫和感器有所不同。阈值被定义为浓度的量的标准误差不与所测试的最低浓度的标准误差重叠。 </li></ol> 4. RNA分离和测序（图2） <ol><li>通过在干冰冷却他们组织disruption.Prepare陪同无RNase杵适合扣帽管紧之前，请准备紧紧贴合无RNA酶的杵。保持关闭，除非积极解剖，以避免过多的冷凝收集管。 </li><li>使用过滤吸引，放在冷的阶段，排序按性别30〜40冷麻醉蚊子（15秒-20℃的冰箱）。地方动物背面向下，抓翅膀不损害味道的附加物。</li><li>使用两对细镊子，解剖来自男性或女性配对labella或跗骨解剖显微镜下，限制包括邻近组织。 注:500配对labella产生大约800-1000毫微克的总mRNA的。 400个人跗节（各5段）产生1,200-1,800纳克总mRNA的。 </li><li>随着1对喘气，钳，轻轻抓住蚊子的长鼻刚好接近labella露出内口针。使用其他对集合镊子，取出labella并添加组织，以冷收集管端。 </li><li>随着一对钳，轻轻抓住蚊子腿的胫骨和跗首段的交界处。使用其他钳子，去除跗节，并添加组织，以冷收集管端。 注:考虑到细胞类型的有用和无用的组织接口。而重要的是要限制本列入邻近组织的，但同样重要的是不能省略期望的组织的部分。该网络最终样本必须准确地代表相关的整个器官。创建与抓钳，这样收集的镊子可以精确解放刮或只抓取所需的组织精确的边界。 </li><li>周期性的，短暂0℃的离心机降速收集管在9000 XG保持在组织管的底部。如果湿气清扫过程中积累的收集管，冷Trizol试剂的添加50μl的覆盖收集的组织。 </li><li>采用防污实验室标志，清楚标示1无RNase1.5毫升扣帽筒每个组织类型的集合。用1.5ml的管架和液氮，冷冻然后研磨组织成细粉（细块，如果在Trizol法）。重复一次。带来的Trizol总体积至1ml和通过涡旋重新悬浮地面组织。 </li><li>加入200微升氯仿调匀。 5分钟后，在室温下，在离心机降速在4℃和11000×g离心15分钟。小心直接添加水层，以基因组DNA过滤柱。降速在11000×g下5分钟。添加等体积的不含RNA酶的70％的乙醇的流通，并用移液管混合。液体转移到RNA的收集列，并继续按照提供的说明RNA提取。 </li><li>量化和评估精密分光光度计总RNA的纯度和进行任何标准的RNA测序方法。 </li></ol> 5.定量RT-PCR RNA测序的确认（图3） <ol><li>无论是在预测序列的丰度和推定化学感受基因功能选择尽可能多的基因尽可能多的动态范围进行比较的相对基因表达水平。 </li><li>设计的引物对为每个目标基因扩增特定100-180碱基对的PCR产物。通过确保每个组的至少一个引物中排除非特异性扩增的gDNA跨越内含子边界。另外，使用DNA酶处理以减少基因组污染。 </li><li>收集如前节所述昆虫组织。计算多少组织被要求提供足够的RNA完成所有QRT-PCR反应。 注:生物一式三份和技术一式三份的反应将提供最大的信心的结果。 </li><li>计算相对基因定量为X 目标 -数（Ct [目标] -CT [参考]），为每个目标基因和平均每个重复，生物和技术。用看家基因（S）正常化生物复制的Ct值，并根Y轴规模相对表达的比较。 </li><li>评估RNA的序列，并使用基于回归的，自举等价方法9，迭代应用，从每个组织的数据的qRT-PCR数据集的相似性。 注:此过程识别可以在周围观察到的RNA-SEQ和定量RT-PCR数据构建的最小的"相似的区域"，并让相关的基因表达由qRT-测PCR技术被认为是统计学相当于相对基因表达的RNA的序列的测定。 </li></ol>

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The authors have nothing to disclose.

的记录动作电位从味觉感受器的最具挑战性的方面是决定哪些响应是"正常的"。当使用单一味觉感器尖记录第一时间为给定的昆虫物种，总数和味觉受体的神经元的敏感性（GRNS）是可能不明。来决定的范围内，化学品，以测试的浓度许多初步录音首先必需的。在这种情况下，我们开始与该单个的GRN响应低浓度DEET（ 图1）的观察。接着，我们研究了同感器内的反应的范围内已知的苦味或厌恶到其他昆虫的化学物质的浓度，并观察到了类似的尖峰形状和振幅（ 图1）。这些观察表明，单一的GRN亚型响应这些厌恶化合物。为了比较，我们测试了已知刺激喂养mosqu含糖化合物itoes，看是否有类似的秒杀发生。我们在这种情况下，观察到一个不同的，较大的尖峰。同样，我们响应于盐刺激观察到的第三，中间大小的尖峰，使总的GRN亚型计数到三个。 昆虫电生理记录准备工作本质上是可变的。靶味觉感受器通常非常小，并且必须被定位在适当的角度与记录电极来访问而不触及玻璃到角质层。建立一个导电桥到味觉神经元，感器的终端孔隙必须打开所含淋巴以建立与记录电极的电解质直接接触。我们已经观察到外来杂物感器开口或者通过堵塞或排出，并从靶感器内干燥的材料。为了避免这些障碍，必须注意的饲养和处理的昆虫，以避免与目标感器接触时服用。除了芝唱损坏，健康测试昆虫，生物因素如年龄，喂养状态下，配合建立控制时，以减少的变异录音应考虑状态和zeitgeber时间。 它可能难以一致地获得一个响应制剂。许多试验可能是必要的，以改善信噪比足以分辨单个尖峰。在上述制剂的实验变化可能导致更好的结果。明智的做法是建立在你的记录设备，强大的响应测试用种常见的昆虫秒杀激发，如蔗糖或奎宁。一旦味觉感受器的响应，这些响应应该是可重复的。时间的刺激之间的最小长度应通过试验确定检查在不同时间约束穗动力学。应避免过度刺激保持GRN健康和防止感器淋巴液退化。在的情况下塔吨响应灵敏度第一刺激后迅速下降，必须获得更多的棉片，提供信心，这些反应是生理上典型的给定的品种。 一些试验化学品的溶解度可以创建困难使得刺激电极，因为有些溶剂可能影响GRN活性，即使在低的终浓度。这些考虑可以通过试验和错误，并用适当的控制来解决。所使用的任何溶剂应加控制电解质和其他的测试解决方案，以确保响应的一致性。固定化小昆虫，从而提供直接访问目标感受器，而不会损坏脆弱的组织，可能需要反复试验。 电的主要限制是所观察到的神经元的反应和潜在的下游行为反应的不幸的分离。因此，有利的是，测试已经B反应的化学物质EEN表现出引起特定的行为反应，以讨论在生态环境的结果的意义。提示录音允许非常准确的检测细胞活性，使得这项技术非常适用于评估转基因动物或参展者的行为表型差异。这些录音还提供关于神经元的反应，如时间和振幅动力学的详细信息，不是依赖于神经元活动的视觉记者系统。在某些情况下，神经反应可以更容易地被量化比行为数据12。 组织收集的RNA序列和定量RT-PCR是简单，但需要不断集中和注重通过冷库和避免多余的水分保持细胞物质。 RNA酶的活动会影响RNA的质量;因此，组织必须只能与清洁的表面遍布的收集和RNA提取。虽然需要定量RT-PCR我的故障排除结果好记录，设计引物和选择的目标和看家基因时，最有可能出现的缺陷在早期。基因可预测地表示均匀间隔的点沿一条线的相对表达水平的是用于比较的RNA序列和定量RT-PCR检测的数据集（ 图3）是有用的。例如，选择的基因的qPCR，显示为0，10，20，30等，分别提供丰富的可预测的顺序RPKM值进行测试。 RNA-SEQ是成功的，在强调低在我们的味觉组织调查表达基因（ 图2）。可靠的基因构建阂。伊蚊 （AaegL1.3，http://aaegypti.vectorbase.org/GetData/Downloads/）和以前的化学感应基因鉴定14取得进球的基因表达相对容易。为了这些目的，昆虫可用基因注解和感兴趣的特定基因家族是有价值的模型系统。确定一个准确的门槛基因表达与背景噪音是投机性的，但确定合理的临界值的方法可用15-17。在这里，我们确定完全信任的RPKM水平和信心下降的基础上的先例9，我们陷害我们的研究结果讨论一个较低的水平RPKM。 在未来，我们希望解剖同时在蚊子和其他重要经济昆虫等化学感受组织，以扩大我们的调解驱虫基因的知识。我们已经建立了几个候选味觉的基因，通过比较基因序列，以品种与现有功能数据13最有可能调解回应厌恶的化合物。我们正在生成蚊子缺少这些个别化学感受基因的转基因株系。我们将使用尖端的录音，以评估对动物的检测已知的驱虫剂，并评估这些突变体的行为反应，以REPE能力，这些基因的作用llents。在该实例中，这些候选基因不显著影响斥，更高的分辨率可能是必要的，以更准确地确定在单个感器或细胞的基因表达或蛋白质的存在。低表达的受体可以是关键指标筛选新颖的驱虫剂，以及普遍表达共受体。不幸的是， 原位杂交和免疫定位已证明非常困难的在昆虫味觉组织18。 一旦我们已经建立了哪些基因介导这些生理反应和行为，我们可以表达这些基因在细胞系统，是适合于候选驱虫剂的高通量筛选。在使用结构模型7也可以是在预测哪些类型的化合物将引起来自特定受体强烈的反应有用硅片的方法。

像DEET，Picaridin，香茅醛和IR3535化合物已被证明有效的驱蚊，包括重要的病媒埃及伊蚊 1,2。我们从具体的味觉感受器相关决定参与蚊虫驱避细胞感觉神经元记录动作电位。再加上表达的基因，在这些组织中的下游测序，我们可以识别以筛选改善斥功能的新的化合物的基因最有可能介导这些细胞的反应。 RNA-SEQ是一个强大的工具，迅速成为标准的跟踪基因表达的时空变化。昆虫化学感受附属物的RNA序列分析和器官已被用来发现几种昆虫物种3-5分子的受体，通过基因6大大提高在常规的基于PCR的基因搜索。昆虫是最多样的动物类，预senting许多机会来研究基因和独特的表型之间的连接。 RNA-SEQ技术可以受雇于任何活昆虫组织。同样地，电生理记录从内部uniporous味觉感受器感觉细胞可以在许多不同的昆虫种类来实现。这两种技术的配对使研究人员能够缩小参与的观察化学感受表型组基因。不同物种会出现特殊的挑战，但可以通知化学感应受体基因和化学感受适应之间的连接。的大小和化学感受感器的形态是可变的，记录动作电位时，以减少噪音，并确定可重复的信号可能需要大量的故障排除。化学感受器官的解剖可能是微不足道的或微妙且耗时的，这取决于形态昆虫和大小。回收的高品质RNA可能需要一些故障排除为好，如避免在某些颜料组织收集。 而通过行为试验证实剂化合物的作用是直接的和翔实的，这种方法是时间密集和广泛的方面的作用机制。电加上RNA-SEQ允许的是什么驱使避税行为的昆虫更具体的分析。一旦化学歧视的"工具箱"已经在昆虫物种鉴定，更具体的尝试，以改善已知的驱虫剂是可能的。受体和相关蛋白在负责这些行为感官细胞可以异源直接化学筛选来表示。此外，分子模型可以预测哪些化学物质会引起强烈的反应，这些受体7。 的一组化学感受组织窄所有活性的基因的快照也可能是在鉴定相似的基因在其他物种有用的。使用序列同源性和表达SImilarities，研究人员可能形成集分子受体最有可能调解反应驱虫剂是昆虫相当有效的。我们提出以下方案，以帮助研究人员在解构昆虫化学感受途径和说服更多的深入到非型号和重要经济昆虫的神经行为学。

<table><tbody><tr><td>Glass capillary</td><td>A-M Systems</td><td>628000</td><td>Standard, 1.5 mm X 0.86 mm, 4&quot;</td></tr><tr><td>Silver wire</td><td>A-M Systems</td><td>7875</td><td>.015&quot; bare</td></tr><tr><td>Tungsten wire</td><td>Alfa Products</td><td>369</td><td>0.127 mm diameter</td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td>11252</td><td>#5SF Inox</td></tr><tr><td>Refridgerated stage</td><td>BioQuip Products</td><td>1429</td><td>Chill Table</td></tr><tr><td>Preamplifier</td><td>Syntech</td><td>Taste Probe</td><td>preamplifier</td></tr><tr><td>Software for electrophysiology</td><td>Syntech</td><td>Autospike</td><td>software for electrophysiology</td></tr><tr><td>TetraMin fish food</td><td>Tetra</td><td>Tropical fish food granules</td><td>fish food ground to fine powder</td></tr><tr><td>TRIzol</td><td>Life Technologies</td><td>15596-026</td><td>RNA isolation reagent</td></tr><tr><td>RNeasy Plus Mini Kit</td><td>Qiagen</td><td>74136</td><td>includes gDNA eliminator and RNeasy spin columns</td></tr><tr><td>Nanodrop spectrophotometer</td><td>Nano Drop Products</td><td>ND-1000</td><td>tabletop spectrometer</td></tr><tr><td>R statistics</td><td>freeware (created by Robert Gentleman and Ross Ihaka)</td><td>www.r-project.org</td><td>Use the lm function of the stats package and the equiv.boot function of the equivalence package in the R computing environment.</td></tr><tr><td>1.5 ml tube rack</td><td>Evergreen</td><td>240-6388-030</td><td>Pour liquid nitrogen into a few empty wells to freeze and grind tissue.</td></tr><tr><td>1.5 ml collection tubes with pestle</td><td>Grainger</td><td>6HAX6</td><td>RNase free</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>11178160</td><td>Spin down frozen tissue to keep at bottom of 1.5 ml tube.</td></tr><tr><td>Primer-BLAST Primer Designing tool</td><td>NCBI</td><td>n/a</td><td></td></tr></tbody></table>

physiological recordings and rna sequencing of the gustatory appendages of the yellow-fever mosquito aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

跟踪记录从阂动作电位。伊蚊味觉感受器（ 图1）证明直接刺激与一系列的化学品的有效性。这种技术可用于通过计数一给定幅度和持续时间的尖峰在一个合理的时间范围内（一般小于500毫秒）来量化反应的任何化学刺激。迹记录必须在给定的实验条件下很容易地再现的。否则，所观察到的生理反应可以表示试验物种的个体之间罕见的表型。 原始RNA-SEQ数据需要被提出作为圆润RPKM前过滤和准确的映射（映射的数量每百万成绩单碱基长度读取读取映射）或FPKM（每百万成绩单碱基长度读片段读取数映射）数值（ 图2）。之间的区别这些两个RNA-SEQ标准化方法已在2开创性作品10,11进行了说明。总mRNA测序的优点之一是能同时检测大量的基因家族的表达的能力。许多基因的表达的视觉呈现一次允许的分子工具集为给定的组织独特的理解。性别，发育阶段或组织类型之间的比较，可以迅速地进行。 生物复制的RNA-SEQ数据集可以通过成本或组织收集的难度的限制。 QRT-PCR提供了用于验证总数的基因表达的小的子集以更低的成本的能力，并且需要更少的源的RNA。并排数据比较（ 图3A）允许更大的信心的RNA序列的结果，因为这两个方法依赖于不同的化学物质用于基因丰度估计。统计等价功能（ 图3B）表明确定性的每种情况下的限制。 <img alt="图1" src="/files/ftp_upload/52088/52088fig1highres.jpg" /> 图1:从阂的唇瓣一个中型感器电生理记录。雌性埃及伊蚊 ，从引工作12修改。跟踪显示响应100毫米氯化钠，100毫米蔗糖，1 mM的奎宁，0.8％避蚊胺，0.8％Picaridin，0.8％，香茅醛和0.8％IR3535。三个尖峰具有不同幅度或形状被标记的a，b或c。分别盐，糖，和苦，:这三个尖峰对应有三个感觉神经元亚型具有不同的敏感性。 <img alt="图2" src="/files/ftp_upload/52088/52088fig2highres.jpg" /> 图2:表通过RNA-SEQ 8组织样品作为测定，从引工作13改性味觉受体基因表达的个体细胞中报告RPKM值每个味觉基因注释，雄性组织（左）和雌性组织（右）。数值四舍五入至最接近的十分之一。热图的颜色强度上限最高RPKM和中点缩放每个基因家族独立。只有一个在这种情况下显示的基因家族。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52088/52088fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52088/52088fig3highres.jpg" /> 图3:对QRT-PCR数据和RNA-SEQ数据，从引工作13改性的比较（A）的横轴的各化学感受基因是由两个数据列来表示。左边的列表示由RNA-SEQ确定的相对表现;化学感受基因RPKM值由该的看家基因Aaeg LASP被分频以产生在该组织中的数值的程度。正确的列代表通过定量RT-PCR确定的相对表现。第一，生物复制的Ct值labella基因使用Aaeg LASP作为标准进行了标准化。其次，每个基因被用来计算相对表达值复制的Ct值（E 目标 -数（Ct [目标] -CT [参考]））。误差棒表示分别计算相对表达值的标准偏差。（B）中的RNA序列和定量RT-PCR检测的数据集在一个单独的分析进行比较。纵轴表示相对基因丰度就证明了RNA的序列数据，具有值1被归因于持家基因Aaeg LASP和所有其他的基因表达被报告相对于这个值。水平轴表示相对基因丰度为预计通过qRT-PCR，用值1被归因于持家基因Aaeg LASP和所有其他的基因表达被报告相对于这个值。红线代表确切的对等。虚线表示&#39;区相似的"为斜率，由统计算法等价9计算的边界。纯黑色线代表的定量RT-PCR等价最小二乘回归到RNA-SEQ定量为12靶基因和Aaeg LASP看家基因，每个圆圈的数据点来表示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52088/52088fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti at JoVE.com

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

使用两种方法来估计基因表达的埃及伊蚊的主要味觉附属物，我们已经确定了一套基因假定存在的潜在的神经元的反应，以苦和排斥的化合物，通过电生理检查确定。

生理录音和黄热病蚊子的味觉附属物RNA测序<em&gt;埃及伊蚊</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

physiological-recordings-rna-sequencing-gustatory-appendages-yellow

Nanyang Technological University

Research

JoVE Journal

Biology

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

8.0K 次观看.  United States Department of Agriculture. 使用两种方法来估计基因表达的埃及伊蚊的主要味觉附属物，我们已经确定了一套基因假定存在的潜在的神经元的反应，以苦和排斥的化合物，通过电生理检查确定。 

视频: Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

从AE肠和唾液腺的解剖。埃及伊蚊蚊

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

生物学

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

免疫与感染

Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.

Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering ...

遗传学

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong preference for human hosts over other warm-blooded animals for blood meals. Olfactory cues play a critical role for mosquitoes as they explore their environment and seek a human host to obtain blood meals, thus transmitting human diseases. Odorant receptors (ORs) expressed in the olfactory sensory neurons are known to be responsible for the interaction of mosquito vectors with human odors. To gain deeper insights into Ae. aegypti&#8217;s olfactory physiology and investigate their interactions with humans at the molecular level, we used an optimized protocol of Xenopus Oocytes heterologous expression to functionally analyze Ae. aegypti odorant receptors in response to human odors. Three example experiments are presented: 1) Cloning and synthesizing cRNAs of ORs and odorant receptor co-receptor&#160;(Orco) from four to six days old Ae. aegypti antennae; 2) Microinjection and expression of ORs and Orco in Xenopus oocytes; and 3) Whole-cell current recording from Xenopus oocytes expressing mosquito ORs/Orco with a two-electrode voltage-clamp. These optimized procedures provide a new way for researchers to investigate human odor reception in Aedes mosquitoes and reveal the underlying mechanisms governing their host-seeking activity at a molecular level.

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human odors.

The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

用于下一代测序的基于磁珠的蚊子DNA提取协议

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

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.

生理录音和黄热病蚊子的味觉附属物RNA测序

摘要

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此视频中的章节

在昆虫单感器记录果蝇按蚊

从AE肠和唾液腺的解剖。埃及伊蚊蚊

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

在伊蚊中喂养和量化动物衍生的血液和人工膳食

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

用于下一代测序的基于磁珠的蚊子DNA提取协议

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药

生理录音和黄热病蚊子的味觉附属物RNA测序

摘要

探索更多视频

此视频中的章节

在昆虫单感器记录果蝇按蚊

从AE肠和唾液腺的解剖。埃及伊蚊蚊

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

用于下一代测序的基于磁珠的蚊子DNA提取协议

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

在伊蚊中喂养和量化动物衍生的血液和人工膳食

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药