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?...

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 border="0" cellpadding="0" cellspacing="0" width="1272">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Name of Material/ Equipment</td>
			<td style="width:367px;">Company</td>
			<td style="width:189px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glass capillary</td>
			<td style="width:367px;">A-M Systems</td>
			<td style="width:189px;">628000</td>
			<td>Standard, 1.5mm X 0.86mm, 4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Silver wire</td>
			<td style="width:367px;">A-M Systems</td>
			<td style="width:189px;">7875</td>
			<td>.015&#34; bare</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tungsten wire</td>
			<td style="width:367px;">Alfa Products</td>
			<td style="width:189px;">369</td>
			<td>0.127mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Fine forceps</td>
			<td style="width:367px;">Fine Science Tools</td>
			<td style="width:189px;">11252</td>
			<td>#5SF Inox</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Refridgerated stage</td>
			<td style="width:367px;">BioQuip Products</td>
			<td style="width:189px;">1429</td>
			<td>Chill Table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Preamplifier</td>
			<td style="width:367px;">Syntech</td>
			<td style="width:189px;">Taste Probe</td>
			<td style="width:467px;">preamplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Software for electrophysiology</td>
			<td style="width:367px;">Syntech</td>
			<td style="width:189px;">Autospike</td>
			<td style="width:467px;">software for electrophysiology</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">TetraMin fish food</td>
			<td style="width:367px;">Tetra</td>
			<td style="width:189px;">Tropical fish food granules</td>
			<td>fish food ground to fine powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy Plus Mini Kit</td>
			<td>Qiagen</td>
			<td style="width:189px;">74136</td>
			<td style="width:467px;">includes gDNA eliminator and RNeasy spin columns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop spectrophotometer</td>
			<td>Nano Drop Products</td>
			<td style="width:189px;">ND-1000</td>
			<td>tabletop spectrometer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:251px;">R statistics</td>
			<td style="width:367px;">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 height="21">
			<td height="21" style="height:21px;width:251px;">1.5ml tube rack</td>
			<td style="width:367px;">Evergreen</td>
			<td style="width:189px;">240-6388-030</td>
			<td>Pour liquid nitrogen into a few empty wells to freeze and grind tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">1.5mL collection tubes with pestle</td>
			<td style="width:367px;">Grainger</td>
			<td style="width:189px;">6HAX6</td>
			<td>RNase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Centrifuge</td>
			<td style="width:367px;">Thermo Scientific</td>
			<td style="width:189px;">11178160</td>
			<td>Spin down frozen tissue to keep at bottom of 1.5 mL tube.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primer-BLAST Primer Designing tool</td>
			<td style="width:367px;">NCBI</td>
			<td style="width:189px;">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-...

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

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

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

建设一个更美好的蚊子：确定基因在冈比亚按蚊和埃及斑蚊蚊子启用疟疾和登革热的电阻

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

科研

生物学

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi介导的基因敲除和<em&gt;在体内</em在成年女性利尿含量<em&gt;埃及伊蚊</em&gt;蚊子

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

的16S核糖体RNA基因的扩增子的新一代测序

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

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, ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

在EML造血前体细胞调节自我更新和分化的关键因素，通过RNA测序分析鉴定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

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 ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

维护伊蚊蚊蚊子感染沃尔巴克

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness parameters, in particular egg production and hatch rates at the individual level, conventional methods have put a substantial burden on investigators due to high labor intensity and laboratory space requirements. Described is a simple method using 24 well tissue culture plate with agarose in each well and digital imaging of each well to determine egg numbers and hatch rates at an individual level with substantially reduced time and space requirements.

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays.

使用 24 个井组织培养板 （EAgaL 板） 改进 了伊蚊 的丰度和生育率测定

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...