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

Nous reconnaissons Heng Lin Yeap, Chris Paton, Petrina Johnson et Clare Doig pour leur contribution au développement de méthodes d’entretien de notre colonie et trois évaluateurs anonymes pour leurs suggestions qui ont contribué à améliorer le manuscrit. Notre recherche est supporté par un programme de subventions et de bourses à l’AAH de la santé nationale et Conseil de recherches médicales et une traduction accordent du Wellcome Trust. NOMINALE est récipiendaire d’une bourse du programme formation de recherche gouvernement australien.

Sang alimentant des moustiques sur des êtres humains a été approuvé par l’Université de Melbourne le Comité éthique humaine (approbation #: 0723847). Tous les bénévoles fournis a informé consentement écrit.
1. larvaire d’élevage
NOTE : Les moustiques sont maintenus à 26 ± 0,5 ° C et 50-70 % d’humidité relative, avec une photopériode de h (lumière : obscurité) 12:12 pour le protocole d’entretien de cette colonie. Ces conditions sont similai...

Selon le protocole présenté ici pour la maintenance de Wolbachia-infectés Ae. aegypti devraient garantir que les moustiques sains d’une qualité constante sont produites pour des expériences et ouvrir le champ communiqués. Contrairement aux autres protocoles qui donner la priorité à la production de grandes quantités de moustiques (voir référence57), les méthodes sont axés sur la maximisation de leur remise en forme, tant au sein des générations en mettant en plac...

Les moustiques Aedes aegypti sont chargés de transmettre certains de l’arbovirus plus importants dans le monde, y compris la Zika, dengue et chikungunya1. Ces virus deviennent une menace croissante pour la santé mondiale comme la généralisation de l' Ae. aegypti dans les tropiques continue à s’étendre de2,3,4. Femelles Ae. aegypti préférentiellement se nourrissent de sang humain5 et donc ont tendance à vivre à proximité de l’homme, en particulier dans les zones urbaines où les populations sont plus denses. Grâce à cette étroite association avec les humains, ils se sont également adaptés pour se reproduire dans des habitats artificiels, y compris les pneus, les pots, les gouttières et les eau réservoirs6,7. AE. aegypti aussi facilement s’adapter à des environnements de laboratoire où elles peuvent être maintenues sans toute exigence particulière après recueillie directement dans le champ, à la différence d’autres espèces dans le genre Aedes la8, 9,10. Leur facilité d’entretien les a vus a étudié largement au laboratoire dans un large éventail de domaines, en fin de compte visant à contrôler les moustiques maladies peut-être transmettre.
Traditionnellement, les arbovirus contrôle s’appuie fortement sur l’utilisation d’insecticides pour réduire les populations de moustiques. Cependant, il y a un intérêt croissant dans les approches où les moustiques modifiés sont élevés en laboratoire et ensuite rejetés dans les populations naturelles. Libéré de moustiques peuvent être modifiés génétiquement11,12,13, biologiquement14,15, par irradiation16, traitement chimique17,18, ou pour l’ensemble des techniques de19 pour réprimer les populations de moustiques ou remplacez-les par des moustiques qui sont réfractaires à arbovirus transmission20.
Wolbachia sont des bactéries qui sont actuellement utilisés comme agent de lutte biologique pour les arbovirus. Plusieurs souches de Wolbachia ont été récemment introduits dans Ae. aegypti expérimentalement à l’aide de microinjection embryonnaires21,22,23,24. Ces souches réduisent la capacité des arbovirus à diffuser et à reproduire dans le moustique, diminuant leur transmission potentielle23,25,26,27,28 . Les infections de Wolbachia sont transmises de la mère à la progéniture, mais certaines souches induisent la stérilité, lorsque les hommes infectés s’accoupler avec les femelles infectées22. Wolbachia-mâles infectés peuvent donc être libérés en grande quantité pour réprimer les populations de moustiques naturels, comme l’a récemment démontrées à l’autre de15,espèces Aedes 29. Toutefois, étant donné que Wolbachia aussi inhiber la transmission arbovirue dans Ae. aegypti, moustiques peuvent être libérés pour remplacer les populations indigènes par les vecteurs les plus pauvres. Ae. aegypti infectés expérimentalement par Wolbachia sont maintenant libérés sur le terrain dans plusieurs pays à l’aide de cette dernière approche14,30,31.
Wolbachia-approches basées pour contrôle arboviru reposent sur une bonne compréhension des interactions entre le Wolbachia, les moustiques et l’environnement. Wolbachia sont présents naturellement dans un large éventail d’insectes, et les souches introduites dans les moustiques sont variés dans leurs effets32. Nouveaux types d’infection de Wolbachia sont introduit dans Ae. aegypti24, il est nécessaire de caractériser chaque souche pour leurs effets sur l’aptitude de moustique, de reproduction et de brouillage arboviru dans un éventail de conditions. Une expérimentation rigoureuse en laboratoire est donc nécessaire d’évaluer le potentiel des souches de Wolbachia réussir dans le domaine.
Rejets de plein champ de Ae. aegypti et les infections de Wolbachia peuvent souvent nécessiter des milliers, des dizaines de milliers de moustiques par zone de libération à être élevés chaque semaine14,30,31. Le succès des versions initiales peuvent être amélioré en libérant des moustiques d’une grande taille afin de maximiser leur fécondité33 et accouplement succès34,35. Les moustiques doivent également être adaptées aux conditions qu'ils vivront dans le champ, élevage de laboratoire cependant à long terme peut causer des changements dans le comportement et la physiologie qui pourrait avoir une incidence champ performance36,37, 38.
Les auteurs décrivent un protocole simple pour l’élevage des Ae. aegypti en laboratoire au moyen de l’équipement de base. Ce protocole est convenable pour les deux sauvage et Wolbachia-infectée de moustiques, laquelle peut nécessitent une attention particulière, comme certaines souches de Wolbachia ont des effets importants sur les moustiques démographiques caractéristiques39, 40. les conditions d’élevage éviter le surpeuplement et la compétition pour la nourriture produire des moustiques d’une taille cohérente, qui est cruciale pour la compétence vectorielle et expériences de remise en forme et veille à ce que les moustiques sont sains pour la libération de champ41 . Nous avons également prendre des précautions pour minimiser l’adaptation de laboratoire et d’endogamie en réduisant les pressions sélectives et veiller à ce que la prochaine génération est prélevé dans une grande piscine au hasard. Cependant, des environnements de laboratoire sont distinctement différentes de conditions sur le terrain, et une maintenance à long terme dans des conditions détendues pourrait réduire l’aptitude des moustiques leur libération dans la zone37,,42,43 . Nous traversons donc femelles de lignes laboratoire récoltés sur le terrain des mâles périodiquement, résultant dans les colonies qui sont génétiquement similaires pour les comparaisons expérimentales et qui sont adaptés à la population de zone cible39. Les méthodes ne requièrent pas de n’importe quel équipement spécialisé et peuvent évoluer vers le haut à l’arrière des dizaines de milliers de personnes par semaine pour les rejets de champ. Le protocole a aussi priorise l’aptitude des moustiques dans et entre les générations, une considération importante pour les insectes destinés à la mise en place dans les populations naturelles. Le protocole est adapté pour la plupart des laboratoires nécessitant une maintenance d' Ae. aegypti, en particulier pour les comparaisons expérimentales où la qualité uniforme de moustiques et narrabilité sur le terrain sont importantes.

<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

Les moustiques Aedes aegypti infectés expérimentalement par Wolbachia sont utilisés dans des programmes pour lutter contre la propagation des arbovirus telles que la dengue, chikungunya et Zika. Wolbachia-moustiques infectés peuvent être rejetées dans le champ soit réduire la taille des populations par le biais de croisements incompatibles ou de transformer les populations de moustiques qui sont réfractaires à la transmission du virus. Pour ces stratégies réussir, les moustiques libérées dans le domaine du laboratoire doivent être compétitives avec les moustiques indigènes. Cependant, maintenir les moustiques en laboratoire peut entraîner consanguinité, dérive génétique et l’adaptation de laboratoire qui peut réduire leur aptitude dans le domaine et peut-être entraîner des résultats d’expériences. Pour tester la pertinence des différentes infections de Wolbachia pour le déploiement sur le terrain, il est nécessaire de maintenir des moustiques dans un environnement de laboratoire contrôlées à travers plusieurs générations. Les auteurs décrivent un protocole simple pour maintenir les moustiques de l’Ae. aegypti dans le laboratoire, qui convient à la fois Wolbachia-moustiques infectés et sauvage. Les méthodes minimiser l’adaptation de laboratoire et mettre en œuvre les croisements pour améliorer la pertinence des expériences aux moustiques de champ. En outre, les colonies sont maintenus dans des conditions optimales afin de maximiser leur ADEQUATION a communiqués de plein champ.

Figure 4 illustre les effets de la nutrition sous-optimaux sur le développement des larves de Ae. aegypti . Quand conteneurs sont équipés de 0,25 mg de nourriture par larves par jour ou moins, le temps de développement augmente pour les mâles et les femelles, et est moins synchrone que dans des contenants fournie avec 0,5 mg de nourriture. Si une alimentation adéquate n’est pas fournie pendant toute la durée du développement larvaire, cela ...

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

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Maintenir les moustiques Aedes aegypti infectés par Wolbachia

Les moustiques Aedes aegypti infectés par Wolbachia sont libérés dans les populations naturelles de supprimer la transmission des arbovirus. Nous décrivons des méthodes arrière Ae. aegypti Wolbachia infections en laboratoire pour des expériences et sortie de terrain, prendre des précautions pour réduire au minimum la sélection et l’adaptation de laboratoire.

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

Wolbachia infection bactérienne chez la drosophile

Recherche

Biologie

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.

Microinjection d&#39;embryons aegypti a. Pour obtenir les moustiques transgéniques

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.

Protocole pour la dengue dans les moustiques (Aedes aegypti) et détermination de phénotype d&#39;infection

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.

Le maintien de Wolbachia dans la cellule sans moyen

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.

Dissection des glandes salivaires de l&#39;intestin moyen et Ae. aegypti

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.

Bâtir un meilleur moustique: l&#39;identification des gènes de résistance Activation fièvre malaria et la dengue chez A. gambiae et A. aegypti

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.

Inactivation génique médiée par ARNi et<em&gt; In Vivo</emEssai diurèse&gt; Femmes adultes<em&gt; Aedes aegypti</em&gt; Les moustiques

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.

Physiologiques Recordings et ARN séquençage des annexes gustatives du Mosquito fièvre jaune<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.

Système de Culture ou organe de corps gras chez Aedes Aegypti, vecteur de Virus Zika

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.

Amélioration de la fécondité et de la fertilité pour Aedes aegypti à l’aide de 24 plaques de culture tissulaire de puits (plaques EAgaL)

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