Name: a bacterial oral feeding assay with antibiotic-treated mosquitoes
Uploaded: 2020-09-12T14:00:00
Description: Watch this Scientific Journal Video about A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes at JoVE.com

这项工作得到了国家自然科学基金（第81902094号，81600497）和湖南省科技计划项目（2019RS1036）的支持。

1. 中古解剖和可培养细菌分离
<ol><li>准备蚊子解剖。<ol>
<li>用吸气器在出现7~9天后收集蚊子。麻醉收集的蚊子，让它们在4°C的温度下3~5分钟，并保持蚊子在冰冷的培养皿中麻醉，直到解剖。</li>
	</ol></li>
	<li>对实验室仪器和蚊子表面进行消毒。<ol>
<li>通过喷洒 75% 乙醇对实验台进行消毒，解剖显微镜、钳子和玻璃幻灯片，以避免被环境中的细菌污染。</li>
		<li>准备一个?...

<ol>
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对宿主-微生物相互作用的研究发现，不同的肠道微生物通过不同的机制影响宿主生理。本文介绍了研究蚊子肠道微生物各自作用的方法，包括解剖蚊虫、栽培肠道细菌、抗生素治疗和重新引入感兴趣的细菌。
要成功进行抗生素治疗，在进行实验时必须考虑以下细节。在该协议中，蚊子使用湿润的棉球进行治疗，用10%蔗糖溶液，包括20单位青霉素和20μg每mL链霉素3天<sup class="xre...

蚊子被认为是人类致病疾病最重要的媒介，传播超过一百种病原体，包括寨卡病毒、登革热病毒和疟原虫1。当蚊子采取血餐来获得营养细胞，他们可以意外地从受感染的宿主通过消化道2摄入病原体。重要的是，在血粉消化和病原体入口中起着关键作用的蚊子中游，它蕴藏着高度动态的微生物群3。
几项研究已经确定实验室饲养和现场收集的蚊子微生物群使用培养依赖方法或细菌测序测定4，5，6。,5,6在各种研究中，包括潘5,7,8,托亚、塞拉蒂亚、克莱布西拉、Elizabethkingia伊丽莎白金氏菌和Enterococcus肠球菌在内的物种通常与蚊子分离。有趣的是，受发育阶段、物种、地理来源和喂养行为的影响，蚊子肠道微生物群在社区多样性和细菌种类中波动剧烈。研究表明，随着肠杆菌物种的迅速扩张和整体多样性的减少，供血Enterobacteriaceae量显著增加,了细菌总负荷。此外，幼虫阶段的蚊子肠道微生物群通常在昆虫在繁殖和闭合过程中发生变质时被根除;因此，新出现的成年蚊子需要重新填充其微生物群4。
古特微生物群在营养吸收、免疫、发育、繁殖和载体能力等各个方面调节昆虫生理学。斧头蚊子幼虫未能发育超过第一星，而细菌口服供应抢救发展，表明蚊子肠道微生物是幼虫发育的关键13，14。,14此外，肠道细菌的耗竭会延缓血餐消化和营养吸收，影响卵母细胞成熟，减少卵巢15。此外，与抗生素治疗的蚊子相比，具有肠道微生物群的蚊子具有更高的免疫反应，与其他病原体的抗菌肽表达不断升高，感染16。在这些研究中，通常口服抗生素来去除泛肠道细菌，然后进行实验，以比较斧头蚊子和蚊子与共生微生物的区别。然而，蚊子中古人蕴藏着各种各样的微生物群落，每种细菌物种对宿主生理机能产生明显的影响。
蚊子微生物群调节病媒能力，具有不同的效应。从登革热流行地区的外地蚊子中分离出的Proteus的殖民化，可产生对登革热病毒感染的增强抗菌肽表达和抵抗力。致病真菌博韦里亚巴西亚纳激活收费和JAK-STAT免疫途径对阿尔博病毒感染17。相比之下，从伊蚊中分离出的真菌Talaromyces通过调节肠道尝试素活性18促进登革热病毒感染。 Aedes aegypti此外，Serratia Marcescens通过一种叫做Sm Enhancin的分Sm泌蛋白促进arbo病毒的传播，这种蛋白质消化蚊子肠道上皮上的粘液层。
该程序为解剖蚊子中口、分离可培养细菌菌落、鉴定细菌种类和通过口服喂养重新引入提供了系统、直观的方法。它提供了具有代表性的用共体细菌、菊花 菌、蚊子卵巢发育和卵巢的供血结果。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td >Sigma</td>
			<td >A2383</td>
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate has been used to prepare adenosine triphosphate (ATP) standard solutions</td>
		</tr>
		<tr >
			<td >Aedes aegypti</td>
			<td></td>
			<td></td>
			<td>Female mosquitoes</td>
		</tr>
		<tr >
			<td >Anticoagulant tube</td>
			<td>BD Vacutainer</td>
			<td>363095</td>
			<td>Collect fresh blood</td>
		</tr>
		<tr >
			<td >Centrifuge tube</td>
			<td>Sangon Biotech</td>
			<td>F601620-0010</td>
			<td>1.5 ml, Natural, Graduated, Sterile</td>
		</tr>
		<tr >
			<td >Cotton balls</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Disposable Tissue Grinding Pestle</td>
			<td>Sangon Biotech</td>
			<td>F619072-0001</td>
			<td>70 mm Long, Conical, Blue, Sterile</td>
		</tr>
		<tr >
			<td >Ethanol absolute</td>
			<td>Paini</td>
			<td></td>
			<td>Dilute it to 75% ethanol</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td>RWD</td>
			<td>F11029</td>
			<td>Dissection</td>
		</tr>
		<tr >
			<td >Hemotek Membrane Feeding System</td>
			<td>Hemotek</td>
			<td></td>
			<td>Components of the feeding system, including&#160; Hemotek temperature controller, feeder-housing assembly, metal feeder assembled.</td>
		</tr>
		<tr >
			<td >Incubator shaker</td>
			<td></td>
			<td>ZQZY-78AN</td>
			<td></td>
		</tr>
		<tr >
			<td >Inoculation Loops</td>
			<td>Sangon Biotech</td>
			<td>F619312-0001</td>
			<td>10 &#956;l, Yellow</td>
		</tr>
		<tr >
			<td >LB Agar Powder</td>
			<td>Sangon Biotech</td>
			<td>A507003</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g&#65307; Agar 15.0 g.</td>
		</tr>
		<tr >
			<td >LB Broth Powder</td>
			<td>Sangon Biotech</td>
			<td>A507002</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g.</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td>Zeiss</td>
			<td>Stemi508</td>
			<td></td>
		</tr>
		<tr >
			<td >Paper cup</td>
			<td></td>
			<td></td>
			<td>Place mosquito</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td>Sangon Biotech</td>
			<td>F104002</td>
			<td>4 inx 125 ft</td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td>Sangon Biotech</td>
			<td>F611203</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin G procaine salt hydrate</td>
			<td>Sangon Biotech</td>
			<td>A606248</td>
			<td>White powder. Soluble in water, soluble in methanol, slightly soluble in water, ethanol</td>
		</tr>
		<tr >
			<td >Single Channal Pipettor</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Streptomycin sulfate</td>
			<td>Sangon Biotech</td>
			<td>A610494</td>
			<td>Streptomycin sulfate is a glucosamine antibiotic that interferes with the synthesis of prokaryotic proteins.</td>
		</tr>
		<tr >
			<td >Sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792</td>
			<td>Soluble in water, ethanol and methanol, slightly soluble in glycerol and pyridine.</td>
		</tr>
		<tr >
			<td >TIANamp Bacteria DNA Kit</td>
			<td>TIANGEN</td>
			<td>DP302</td>
			<td>Extract DNA&#160;</td>
		</tr>
		<tr >
			<td >Utility Fabric-Mosquito Netting White</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vortex mixer</td>
			<td>Scintic Industries</td>
			<td>S1-0246</td>
			<td></td>
		</tr>
		<tr >
			<td >1.5ml EP tube</td>
			<td>Sangon Biotech</td>
			<td>F600620</td>
			<td></td>
		</tr>
		<tr >
			<td >10X PBS buffer</td>
			<td>Sangon Biotech</td>
			<td>E607016</td>
			<td>This product is a 10X solution. Please dilute it 10 times before use. The pH value is 7.4.</td>
		</tr>
	</tbody>
</table>

a bacterial oral feeding assay with antibiotic-treated mosquitoes

蚊子中游人具有高度动态的微生物群，影响宿主的新陈代谢、繁殖、健身和病媒能力。已开展研究，以调查肠道微生物作为一个整体的影响;然而，不同的微生物会对宿主产生不同的影响。本文提供了研究每种特定蚊帐微生物作用和潜在机理的方法。
该协议包含两个部分。第一部分介绍如何解剖蚊子中，分离可培养的细菌菌落，并识别细菌物种。第二部分提供了产生抗生素处理的蚊子的程序，并重新引入一种特定的细菌物种。

用抗生素治疗和没有抗生素治疗的蚊子中游被提取出来进行DNA提取，用通用细菌底剂进行qPCR。 图1 显示了对照组和抗生素治疗组中细菌16S rRNA的表达。结果表明，约98%的肠道细菌已被去除，青霉素和链霉素的肠道灭菌工作成功。
通过所述方法，分离和鉴定细菌菌株。 C. 性消化是 一种非感染性、氧化酶阳性的克阴性有氧杆菌 ，属于菊花菌。 图...

Watch this Scientific Journal Video about A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes at JoVE.com

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

抗生素处理蚊子的细菌口服喂养测定

本文提出了一个协议，研究单个蚊子肠道细菌的影响，包括隔离和识别蚊子中生可培养微生物，抗生素消耗蚊子肠道细菌，并重新引入一个特定的细菌物种。

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

蚊子中，有一个复杂的微生物群落，影响宿主的新陈代谢、繁殖、健身和兽医能力。与每个肠道微生物是一个不同的效果。本视频介绍了研究每种细菌菌株或宿主生理学各自作用的过程。为了获得肠道细菌菌落的培养，在无菌条件下解剖蚊子中肠。地面解剖中，血清，并蔓延到阿加板。从板块中挑选单个菌落，通过16个SRNA基因序列识别细菌物种。连续三天用含有抗生素的COD Embos喂养蚊子，以去除肠道微生物群。抗生素治疗的有效性可以通过在随后的极端分子之前解剖无菌蚊子的中游，以进行聚居形成单位测定来证实。然后重新引入以前通过口服喂养检测从蚊子肠道分离的特定细菌物种。通过比较未经治疗的蚊子、抗生素治疗的蚊子和蚊子重新引入特定的约束，可以评估细菌对宿主生理的影响。中古特解剖和可培养细菌分离。首先，用吸气器将蚊子收集在笼子里，并在收集箱中将蚊子细胞。在4度的温度下对蚊子进行麻醉，三到五分钟。将蚊子放在冰冷的培养皿中，让蚊子麻醉。通过喷洒 75% 乙醇对长凳进行消毒、解剖显微镜和钳子，以避免被环境中的细菌污染。表面消毒蚊子通过录音和摇动他们在75%乙醇三分钟，并用PBS缓冲液冲洗两次。将蚊子单独分在无菌玻璃幻灯片上，该幻灯片包含您的 PBS 滴。小心地用钳子切除蚊子的腿、翅膀和头部。夹住蚊子的胸部和腹部的最后一部分被钳子和拉开，以隔离消化道。使用钳子从消化道上取出作物和麦芽管。作物在摄入糖水后，位于中高和凸起的前部。马尔皮吉安管是一组细长的排泄管在中古和后结的交界处。将解剖的化妆品放入 EP 管中，使用 200 微升无菌 PBS 缓冲液，并在干净的长凳中用无菌研磨牧师研磨。谷物加载同质到三个节奏错觉在 50 微升每个错觉到 Lb 阿加板和绝望的板。将板孵育为37度一至两天，直到单菌落可行。将单个菌落放入含有 50 毫升磅胸罩介质的 150 毫升圆锥形烧瓶中。一夜之间在37度摇动细菌。物种鉴定。通过细菌基因组DNA构建试剂盒提取TOTO DNA，通过PCR扩增16SRDNA。通过安布罗斯凝胶电泳和驱动回收套件从 PCR 产品中恢复和纯化 DNA 片段。对纯化的DNA片段进行DNA测序，以获得细菌链序列。运行爆炸搜索查询16 SRNA基因序列，识别细菌对细菌和古和数据库。对物种级别的识别定义为大于或等于 99%16 SRDNA 序列相似性，以最接近的团队库条目。分离物被分配到相应的属时为16 SRDNA序列，相似度小于99%，大于或等于95%的抗生素治疗和细菌再引入。称量所需的蔗糖青霉素和链霉素，为我们准备10%蔗糖溶液，包括尝试20单位青霉素和20微克链霉素每毫升。将棉球渗入含有抗生素的蔗糖溶液中，放在含有蚊子的纸杯顶部。用10厘米的培养皿盖住棉球，防止水水蒸发。更换棉球两天，连续三天喂蚊子。蚊子分为两组。第一组被放置在一个蚊子杯没有任何治疗，任何棉球医生与10%所以交叉每天给予。第二组接受抗生素治疗，服务三天。在每组，谢尔盖蚊子被解剖。Mika被提取用于总DNA提取，QPCR使用通用细菌底剂进行，图一显示了对照组和抗生素治疗组中16个SRNA的表达。结果表明，青霉素和链霉素的成本或实现是成功的。准备细菌溶液，用分光光度计测量病毒中的细菌溶液，将一个OT细菌悬浮液加入EP管。在5000 RCA下离心5分钟，在四度下丢弃上流水线，用无菌PBS缓冲液清洗细菌托盘尝试。用 200 微升无菌 PBS 缓冲液重新悬浮细菌托盘，添加 600 微升、10%蔗糖溶液、200 微升 ATP 和 200 微升细菌悬浮到新的 AP 管中并混合好。或者，细菌可以重新悬浮在热中激活你的血液。加热活性血液的准备。用抗凝血管凝固新鲜血液，服用两到三毫升新鲜血液。在1000度离心10分钟，在4度分离血浆和血细胞。将等离子体收集到新的 EP 管中，并在 56 度激活一小时。用无菌的 PBS 缓冲液洗净爆炸屋。用加热激活等离子体冲洗悬挂的爆破室。组装膜和进料系统。将薄膜病拉到最大。用塑料环和工作场所的副膜固定，以避免泄漏缓慢地将准备好的细菌溶液添加到进料装置中，添加到最大钱包中。为了避免气泡从一个方向下降，并密封进纸器单元。连接电源。等待，直到温度达到 37 度，在喂食器上安装带细菌溶液的送料器单元。在喂养细菌之前，蚊子应停止24小时代谢抗生素。对于用蚊子在纸杯上的饲料单元，则允许喂食90分钟。可以挑选完全受鼓励的蚊子作进一步研究。代表性结果。图二显示每只蚊子的平均荣誉。在绘制了抗生素喂养后，我们看到了消化不良。图三显示了一个变异发展相关基因的表达24小时后的血液餐含有C性消化。结果表明，对照组和褪色组的卵子产量无显著变化。感谢您对细菌或抗生素治疗蚊子的喂养文章感兴趣。

a-bacterial-oral-feeding-assay-with-antibiotic-treated-mosquitoes

Research

JoVE Journal

Biology

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

注射的。 stephensi胚胎生成抗药性疟疾的蚊子

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

科研

生物学

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

防止疟疾的传播和使用基因改造的蚊子登革热

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

口腔感染杆状病毒鳞翅目幼虫的协议

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

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 ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

高脂饮食范式斑马鱼幼体：饲养，实时成像，和食物摄入的量化

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

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, ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

果蝇的口腔细菌感染和脱落

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

新型杀虫化学对蚊子毒性测试规程

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.