Name: maintaining aedes aegypti mosquitoes infected with wolbachia
Uploaded: 2017-08-14T15:00:00
Description: Watch this Scientific Journal Video about Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia at JoVE.com

Heng Lin Yeap, Chris Paton, Petrina Johnson ve Clare Doig bizim koloni bakım yöntemleri gelişmesine katkılarından dolayı ve el yazması geliştirmeye yardımcı olan öneriler için üç adsız yorumcular anıyoruz. Bizim araştırma programı hibe ve dostluk tarafından AAH için Ulusal Sağlık desteklenir ve tıbbi araştırma Konseyi ve bir çeviri Wellcome Trust verin. PAR bir Avustralya hükümeti araştırma eğitim programı bursu bir alıcı var.

Sivrisinek insan denekler üzerinde kan besleme University of Melbourne insan Etik Komitesi tarafından onaylanmış (onay #: 0723847). Tüm gönüllüler sağlanan yazılı izni haberdar.
1. larva yetiştirme
Not: Sivrisinekler 26 ± 0,5 ° C ile 50-%70 bağıl nem, 12:12 h (ışık: karanlık) photoperiod bu koloni bakım protokol ile düzenlenen. Bu koşullar Ae. aegypti hayatta kalma ve gelişme44,45</s...

Burada Wolbachiabakım için sunulan protokol sonrası-virüslü Ae. aegypti sağlamak tutarlı bir kalite sağlıklı sivrisinekler deneyler için üretilen ve açık alan bültenleri. (Bkz: başvuru57) sivrisinek miktarda üretim öncelik diğer iletişim kuralları aksine, onların zindelik, hem rahat yetiştirme koşulları uygulayarak ve arasında nesiller içinde en üst düzeye çıkarma yöntemleri odaklı akraba evliliği, seçim ve laboratuvar uyum en aza nesiller. Bu i...

Aedes aegypti sivrisinek Dang, Zika ve chikungunya1de dahil olmak üzere dünyanın en önemli arboviruses bazıları iletmek için sorumludur. 2,3,4genişletmek Ae. aegypti tropikal yaygın dağıtım devam ederken bu virüsler küresel sağlık için artan bir tehdit hale gelmektedir. Kadın Ae. aegypti tercihen insan kanı5 besleme ve böylece insanlar, özellikle kentsel alanlarda nüfusun en yoğun olduğu yakın yaşamak eğilimindedir. İnsanlar ile bu yakın ilişki yoluyla onlar da lastikleri, tencere, oluklar ve su tankları6,7de dahil olmak üzere, yapay yaşam alanları içinde üremek için adapte olması. AE. aegypti da kolayca nerede onlar herhangi bir özel gereksinimleri olmadan doğrudan alanından farklı olarak bazı diğer türler Aedes cins8toplanan sonra korunabilir laboratuvar ortamlarına uyum, 9,10. Bakım kolaylığı onları geniş alanları geniş bir laboratuvarda Okulu'nu gördü, sonuçta hastalıkları sivrisinek kontrolü amaçlayan iletmek.
Geleneksel olarak, arboviral denetim ağır sivrisinek örneğin alınma olasılığını azaltmak için böcek öldürücüler kullanımına dayanıyor. Ancak, değiştirilmiş sivrisinek nereye laboratuvarda yetiştirilen ve sonra doğal nüfus serbest yaklaşımlar artan ilgi vardır. Yayımlanan sivrisinekler11,12,13, biyolojik olarak14,15, ışınlama16, kimyasal arıtma17,18genetik olarak değiştirilmiş, ya da sivrisinek nüfus bastırmak veya bunların yerine arboviral iletim20' ye ateşe dayanıklı sivrisinekler teknikleri-19 ile birlikte.
Wolbachia olan bir biyolojik mücadele ajanı olarak arboviruses. için kullanılmakta olan bakteri Birkaç soy-in Wolbachia son zamanlarda deneysel olarak embriyonik mikroenjeksiyon21,22,23,24kullanarak Ae. aegypti tanıtıldı. Bu suşların yaymak ve onların iletim potansiyel23,25,26,27,28 azalan sivrisinek içinde çoğaltmak için arboviruses kapasitesini azaltmak . Ancak hastalık bulaşmamış tek kadın22ile enfekte erkek dostum zaman belirli suşları kısırlık neden Wolbachia enfeksiyonlar çoluk, anneden iletilir. Wolbachia-virüslü erkekler bu nedenle doğal sivrisinek nüfus, son diğer Aedes türler15,29gösterdi bastırmak için büyük miktarlarda yayınlanacak. Wolbachia da inhibe bu yana arboviral Ae. aegyptiiletimde, ancak, sivrisinekler de yerli nüfus ile yoksul vektörel çizimler yerine yayınlanacak. Deneysel olarak Wolbachia ile enfekte Ae. aegypti are şimdi serbest bırakmak içine alan birçok ülkede bu ikinci yaklaşımı14,30,31kullanarak.
Wolbachia-arboviral denetimi için tabanlı yaklaşımlar itimat Wolbachia, Sivrisinek ve çevre arasındaki etkileşimler ses bir anlayış. Wolbachia ortaya doğal olarak çok çeşitli böcekler ve sivrisinekler tanıttı suşları onların etkileri32çeşitlidir. Yeni Wolbachia enfeksiyon türü Ae. aegypti24tanıtıldı gibi sivrisinek fitness, üreme ve koşulları bir dizi altında arboviral girişim üzerindeki etkileri için her zor karakterize etmek gereklidir. Titiz deney Laboratuvarı bu nedenle alanında başarılı olmak Wolbachia suşları potansiyeli değerlendirmek için gereklidir.
Ae. aegypti açık alan bültenleri Wolbachia enfeksiyonları ile kez sivrisinek olmak serbest bölge başına, on binlerce binlerce her hafta14,30,31yetiştirilen gerektirebilir. İlk bültenleri başarısı sivrisinekler onların verimlilik33 ve çiftleşme başarı34,35en üst düzeye çıkarmak için büyük bir boyutta serbest bırakarak geliştirilebilir. Sivrisinekler de alanında, ancak uzun vadeli laboratuvar yetiştirme değişiklikler davranış ve alan performans36,37etkileyen Fizyoloji neden olabilir yaşayacaksınız koşullarına adapte edilmelidir, 38.
Biz Ae. aegypti temel ekipman kullanarak laboratuvar yetiştirme için basit bir protokol tanımlamak. Bu iletişim kuralı için her iki vahşi türü uygundur ve Wolbachia-enfekte sivrisinekler, ikincisi olan gerektirebilir özel ilgi gibi bazı Wolbachia suşları sivrisinek hayat-Tarih özellikleri39, üzerinde önemli etkileri 40. aşırı kalabalık ve vektör yetkinlik ve fitness deneyler için önemlidir ve sivrisinekler için alan serbest bırakmak41 sağlıklı olmasını sağlar tutarlı bir boyuta sivrisinekler üretmek gıda için rekabet yetiştirme durumlardan kaçınmak . Biz laboratuvar adaptasyon ve akraba evliliği seçici baskıların azaltılması ile en aza indirmek önlemler almak ve yeni nesil sağlamak büyük, rasgele havuzundan örneklenir. Ancak, laboratuvar ortamları alan koşullarından farklı ve rahat koşullarda uzun vadeli bakım yayın içine alan37,42,43 üzerine sivrisinek fitness azaltabilir . Bu nedenle kadın alan toplanan erkeklerin periyodik olarak, laboratuvar satırlarından, deneysel karşılaştırmalar için genetik olarak benzeyen, ve için hedef alan nüfus39adapte olan koloniler sonuçlanan geçeriz. Yöntemleri herhangi bir özel ekipman gerektirmez ve arka on binlerce her hafta alan sürümleri için bireylerin ölçeklendirilebilir. Protokol ayrıca fitness kuruluş doğal nüfus için mukadder böcekler için önemli bir göz sivrisinek içinde ve nesiller arasında öncelik. Protokol sivrisinekler ve relatability alanı için tutarlı bir kalite nerede önemli özellikle deneysel karşılaştırmalar için Ae. aegypti, bakım gerektiren çoğu laboratuarlar için uygundur.

<table width="1661">
	<tbody>
		<tr height="24">
			<td height="24" style="width:287px;height:24px;">Wild type Aedes aegypti</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td style="width:1087px;">Collected from field locations in Queensland, Australia, see Yeap and others39 for details</td>
		</tr>
		<tr height="24">
			<td height="24" style="width:287px;height:24px;">w Mel-infected Aedes aegypti</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Provided by Monash University. Refer to Walker and others23 for information on the strain</td>
		</tr>
		<tr height="24">
			<td height="24" style="width:287px;height:24px;">w AlbB-infected Aedes aegypti</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Provided by Monash University. Refer to Xi and others21 for information on the strain</td>
		</tr>
		<tr height="24">
			<td height="24" style="width:287px;height:24px;">w MelPop-infected Aedes aegypti</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Provided by Monash University. Refer to McMeniman and others22 for information on the strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Instant dried yeast</td>
			<td style="width:168px;">Lowan</td>
			<td style="width:119px;"></td>
			<td>Stimulates egg hatching. Found in general grocery stores. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">5 L plastic tub</td>
			<td style="width:168px;">Quadrant</td>
			<td style="width:119px;">Q110950</td>
			<td>Used for hatching and rearing larvae. Other products may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Fish Food (Tetramin Tropical Tablets)</td>
			<td style="width:168px;">Tetra</td>
			<td style="width:119px;">16152</td>
			<td>Provided to larvae as a source of food. Web address: https://www.amazon.com/Tetra-16152-TetraMin-Tropical-10-93-Ounce/dp/B00025Z6SE</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Plastic containers</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Used for rearing larvae. Any plastic container above 500 mL should be suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Glass pipette</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Used for transferring larvae and pupae between containers. Web address: https://www.aliexpress.com/item/10Pcs-Durable-Long-Glass-Experiment-Medical-Pipette-Dropper-Transfer-Pipette-Lab-Supplies-With-Red-Rubber-Cap/32704471109.html?spm=2114.40010308.4.2.py4Kez</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Clicker counter</td>
			<td style="width:168px;">RS Pro</td>
			<td style="width:119px;">710-5212</td>
			<td>Used to assist in the counting of larvae, pupae and eggs. Web address: http://au.rs-online.com/web/p/products/7105212/?grossPrice=Y</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Rearing trays</td>
			<td style="width:168px;">Gratnells</td>
			<td style="width:119px;"></td>
			<td>Used for rearing larvae. Web address: http://www.gratnells.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Nylon mesh</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Used to transfer larvae and pupae to containers of fresh water. Other brands may be used. Web address: https://www.spotlightstores.com/fabrics-yarn/specialty-apparel-fabrics/nettings-tulles/nylon-netting/p/BP80046941001-white</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Cages</td>
			<td style="width:168px;">BugDorm</td>
			<td>DP1000</td>
			<td>Houses adult mosquitoes. Alternative products may be used. Web address: http://bugdorm.megaview.com.tw/bugdorm-1-insect-rearing-cage-30x30x30-cm-pack-of-one-p-29.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">35 mL plastic cup</td>
			<td style="width:168px;">Huhtamaki</td>
			<td style="width:119px;">AA272225</td>
			<td>Used to provide water or sucrose to adult mosquitoes. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">35 mL plastic cup lid</td>
			<td style="width:168px;">Huhtamaki</td>
			<td>GB030005</td>
			<td>Used to provide sucrose to adult mosquitoes. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Cotton wool</td>
			<td style="width:168px;">Cutisoft</td>
			<td style="width:119px;">71841-13</td>
			<td>Moist cotton wool is provided as a source of water to adults. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">White Sugar</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Provided as a source of sugar to adult mosquitoes. Found in general grocery stores</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Rope</td>
			<td style="width:168px;">M Recht Accessories</td>
			<td>C323C/W</td>
			<td>Used to provide sucrose solution to adults. Other brands may be used. Web address: https://mrecht.com.au/haberdashery/braids-cords-and-tapes/cords/plaited-cord/cotton/</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Plastic cup (large)</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>Used as an oviposition container. Any plastic cup that holds 100 mL of water should be suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Sandpaper</td>
			<td style="width:168px;">Norton Master Painters</td>
			<td>CE015962</td>
			<td>Provided as an oviposition substrate. Alternative products may be used, but we use this brand because it is relatively odorless. Lighter colors are used for contrast with eggs. Web address: https://www.bolt.com.au/115mm-36m-master-painters-bulk-roll-p80-medium-p-9396.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Filter paper</td>
			<td style="width:168px;">Whatman</td>
			<td style="width:119px;">1001-150</td>
			<td>Used as an alternative oviposition substrate. Other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:287px;height:21px;">Latex gloves</td>
			<td style="width:168px;">SemperGuard</td>
			<td>Z560979</td>
			<td>Prevents mosquito bites on hands when blood feeding. Other brands may be used. Web address: http://www.sempermed.com/en/products/detail/semperguardR_latex_puderfrei_innercoated/</td>
		</tr>
	</tbody>
</table>

maintaining aedes aegypti mosquitoes infected with wolbachia

Deneysel olarak Wolbachia ile enfekte aedes aegypti sivrisinek arboviruses Dang, chikungunya ve Zika gibi yayılmasını kontrol programlarında kullanılmaktadır. Wolbachia-virüslü sivrisinekler de nüfus boyutları arasında uyumsuz matings azaltmak veya virüs iletim için ateşe dayanıklı sivrisinek ile nüfus dönüştürmek için alana piyasaya sürülmelidir. Bu stratejiler başarılı olmak laboratuvar alanından içine serbest sivrisinek yerli sivrisinek ile rekabetçi olmalı. Ancak, sivrisinekler laboratuvarda Bakımı akraba evliliği, genetik sürüklenme ve onların zindelik alanını azaltabilir ve deneylerin sonuçlarını yıkmak laboratuvar adaptasyon neden olabilir. Farklı Wolbachia enfeksiyonlar dağıtımı için uygunluğu sahada test için sivrisinek kontrollü laboratuvar ortamında birden çok nesiller boyunca korumak gereklidir. Her iki Wolbachiaiçin uygundur laboratuvarında Ae. aegypti sivrisinek korumak için basit bir protokol tanımlayan-virüslü ve vahşi-türü sivrisinekler. Yöntemler laboratuvar adaptasyon en aza indirmek ve deneyler alaka alan sivrisinekler için artırın için outcrossing uygulamak. Ayrıca, koloniler onların zindelik açık alan sürümleri için en üst düzeye çıkarmak için en uygun koşullarda korunur.

Şekil 4 Ae. aegypti larva gelişimine suboptimal beslenmenin etkisi gösterir. Ne zaman kapsayıcı gıda larva günlük başına 0,25 mg ile sağlanır veya daha az, hem kadın ve erkek için Geliştirme süresini artırır, ve daha az kaplar içinde zaman uyumlu gıda 0.5 mg ile sağlanır. Yeterli gıda larva geliştirme süresi boyunca sağlanmazsa, bu bakım planı üzerinde olumsuz bir etkisi olabilir. Yavaş gelişen bireylerin karşı seçil...

Watch this Scientific Journal Video about Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia at JoVE.com

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti sivrisinek enfekte Wolbachia ile bakımını yapma

Wolbachia ile enfekte aedes aegypti sivrisinek arboviruses iletimini bastırmak için doğal nüfus serbest edilmektedir. Biz laboratuvar adaptasyon ve seçimi en aza indirmek önlemler alarak deneyler ve alan açıklaması, arka Ae. aegypti laboratuvarda Wolbachia enfeksiyonları ile yöntemleri açıklanmaktadır.

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

The overall goal of this protocol is to describe the maintenance of the Aedes aegypti mosquitoes infected with Wolbachia. These mosquitoes are being released around the world to suppress the transmission of dengue and other viruses. Our protocol for rearing Aedes aegypti mosquitoes can be used by any researchers who are interested in using mosquitoes for experiments or disease control programs.This method can also be used for rearing genetically modified mosquitoes for mosquito population control programs, including those that involve sterile insect release. The principles involved in this approach can also be applied to the rearing of other disease vector species. For this protocol, house the mosquitoes at approximately 26 degrees Celsius and at 50 to 70%relative humidity with an equatorial photo period.If they are reared at higher temperatures, the Wolbachia infections could be lost, so do not overheat the colonies. Maintain around 500 mosquitoes per population to minimize inbreeding. Using this protocol with adequate nutrition will yield 28 day generations.Begin with eggs on a substrate. Submerge them in trays containing three liters of water with about 300 milligrams of fish food and a few grains of active, dry yeast to induce hatching. One day after hatching, use a glass pipet to transfer approximately 500 larvae to trays containing four liters of water and fresh fish food.Check the trays daily to ensure that the larvae have sufficient food. Add about 600 milligrams of fish food every other day to maintain an optimal food supply. Overfeeding can result in water clouding and thus mortality, so replace the water as needed.If nutrition and temperatures are maintained correctly, the larvae will pupate five to seven days after hatching. And adults will emerge approximately two days later. Seven days after hatching, pour the entire contents of the tray through a fine mesh.Retain some of the filtered water in a five liter container for ova position in the same colony. Next, invert the mesh, and dip it into a plastic container with 200 milliliters of water to transfer the pupae. Add the appropriate amount of food to feed any remaining larvae.Now, set up an adult emergence cage with two cups of 10%sucrose solution and two cups of moist cotton balls to prevent desiccation. If the pupae do not need to be sorted by sex, transfer the container of pupae into a cage, and leave the lid slightly ajar for the adults to escape and enter the cage. After all of the adults have emerged, remove the container from the cage.Do not select against slow developers by taking the container out sooner. To collect virgin mosquitoes, transfer the pupae from the larval rearing trays to plastic containers with 200 milliliters of water every 24 hours. Sort the sexes by size.Females are larger than males. And transfer them to separate containers. Keep the lids closed on the containers so the adults do not escape.Before releasing the emerging adults into their cages, confirm their sex. Incorrectly sexed adults must be removed using an aspirator within 24 hours of emergence while they are still sexually immature. To keep the Wolbachia infected colonies similar to a natural population, cross the infected females from the lab to uninfected wild males.Mature the males and females separately for two days. And then aspirate the females into the male cage. Ideally, set up a one to one sex ratio with a population size of around 500.Proceed to outcross the mosquitoes for at least three consecutive generations. At least three days after the last adults emerge, and before they are two weeks old, blood feed the colony before excessive female mortality occurs. This is particularly important for mosquitoes with Wolbachia infections that shorten lifespan.The sugar cups should be removed the day before. Give the mosquitoes access to a forearm, and wear a latex glove to protect the hand and wrist. Most females should be fed within five minutes.But it can take up to 15 minutes. Once all the females are visibly engorged, remove the forearm, and replace the sugar cups. Two days after blood feeding, place two plastic cups filled with water and lined with a partially submerged strip of sandpaper into the cage.Then, remove any other water source except for the sugar water. The females will oviposit on the sandpaper just above the water line from three days after blood feeding. Collect and replace the sandpaper daily until no more eggs are laid.If more eggs are needed, blood feeding can be performed at weekly intervals. Partially dry the sandpaper with about 30 seconds of gentle blotting using a paper towel. Do not dislodge the eggs.Then, wrap the strips in a sheet of dry paper towel, and place it into a sealable plastic bag. Three days post collection, inspect the eggs under a dissecting microscope. If the sandpaper is too wet, the eggs may hatch before being submerged in water.But if it dried too harshly, the eggs may collapse. The eggs are ready to hatch three days after collection. Submerge all the eggs from each colony into the same container of water to produce a large pool of individuals for the next generation.Generally, eggs can be stored in a sealed container with a humidity greater than 80%at 20 degrees Celsius for several months. But some Wolbachia infections reduce viability, so these eggs should be hatched within a week of collection. When larvae containers were provided with 0.25 milligrams of food per larva per day or less, the development time increased for both sexes and was less synchronous than in containers provided with more food.If adequate food is not provided throughout the duration of development, this will negatively impact colony maintenance. Slowly developing individuals might be selected against, and blood feeding may be delayed. There may even be increased adult mortality before reproduction.Sub-optimal nutrition affected adult morphology. Wing lengths decreased substantially and correlated to nutrition levels. Uniform body sizes are important to experimental comparisons as body size is positively associated with fecundity, and large mosquitoes should be more fit under field conditions.After watching this video, you should have a good understanding of how to maintain Aedes aegypti mosquitoes infected with Wolbachia. Following our protocol should ensure that healthy mosquitoes of a consistent quality are produced. These mosquitoes can then be released into the field for disease control programs or be used for experiments.When working with Wolbachia infected mosquitoes, it is important to monitor their infection status to ensure that the colonies maintain their Wolbachia infections.

maintaining-aedes-aegypti-mosquitoes-infected-with-wolbachia

Research

JoVE Journal

Biology

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Wolbachia Bacterial Infection in Drosophila

Drosophila Wolbachia Bakteriyel Enfeksiyon

Biyoloji

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

Transgenik Sivrisinekler Edinme A. aegypti Embriyolar Mikroenjeksiyon

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Dang Enfeksiyonlar Sivrisinekler (A. aegypti) ve Enfeksiyon Fenotip belirlenmesi için protokol

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

Hücre Orta Wolbachia bakımı

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

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.

Ae midgut ve Tükürük Bezlerinin Diseksiyon. aegypti Sivrisinekler

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

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.

Bina daha iyi Sivrisinek: Sıtma ve Dang Ateşi Direnç A. gambiae ve A. aegypti Sivrisinekler etkinleştirilmesi Genler belirlenmesi

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

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-aracılı Gene Nakavt ve<em&gt; In Vivo</emYetişkin Kadınlar&gt; diürez Testi<em&gt; Aedes aegypti</em&gt; Sivrisinekler

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

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.

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

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Fizyolojik Kayıtlar ve Sarı-ateş Sivrisinek Tat Çıkıntılara RNA Sıralama<em&gt; Aedes aegypti</em

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

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.

Yağ vücut Organ kültür sistemi içinde Aedes Aegypti, bir vektör Zika virüs

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 ...

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.

24 Kuyu Doku Kültürü Plakası (EAgaL Plakaları) kullanılarak Aedes aegypti için Geliştirilmiş Doğurganlık ve Doğurganlık Tahlilleri

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