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

Riconosciamo Heng Lin Yeap, Chris Paton, Petrina Johnson e Clare Doig per il loro contributo allo sviluppo dei nostri metodi di manutenzione di Colonia e tre utenti anonimi per i suggerimenti che ci ha aiutato a migliorare il manoscritto. La nostra ricerca è supportata da un programma grant e la fellowship di AAH dal National Health e il Medical Research Council e una traduzione concedere dal Wellcome Trust. PAR è un destinatario di un australiano governo ricerca formazione programma di borsa di studio.

Alimentazione di sangue di zanzare su soggetti umani è stato approvato dall'Università di Melbourne Comitato etica umana (approvazione n.: 0723847). Tutti i volontari forniti informato consenso scritto.
1. larvale allevamento
Nota: Le zanzare sono tenute presso 26 ± 0,5 ° C e 50-70% di umidità relativa, con un fotoperiodo di 12:12 h (luce: scuro) per questo protocollo di manutenzione di Colonia. Queste condizioni sono simili alle condizioni climatiche media a Cai...

Seguendo il protocollo presentato qui per la manutenzione di Wolbachia-infetti Ae. aegypti dovrebbero assicurare che le zanzare sane di una qualità costante sono prodotte per esperimenti e aprire campo releases. A differenza di altri protocolli che privilegiano la produzione di massa quantità di zanzare (Vedi riferimento57), i metodi si concentrano su come massimizzare la loro idoneità, sia all'interno di generazioni implementando rilassate condizioni di allevamento e da altra...

Le zanzare Aedes aegypti sono responsabili della trasmissione di alcune dell'arbovirus più importanti del mondo, tra cui la dengue, Zika e chikungunya1. Questi virus stanno diventando una crescente minaccia per la salute globale, come la distribuzione capillare di Ae. aegypti nei tropici continua ad espandersi2,3,4. Femmina Ae. aegypti preferenzialmente si nutrono di sangue umano5 e quindi tendono a vivere in prossimità di esseri umani, soprattutto nelle aree urbane, dove le popolazioni sono più densi. Attraverso questa stretta associazione con gli esseri umani si sono adattati anche per allevare in habitat artificiali, compresi i pneumatici, vasi, grondaie e serbatoi acqua6,7. AE. aegypti anche facilmente adattarsi ad ambienti di laboratorio dove può essere mantenute senza requisiti speciali dopo essere raccolte direttamente dal campo, a differenza di alcune altre specie nel genere Aedes 8, 9,10. La facilità di manutenzione ha visto loro ampiamente studiato in laboratorio in una vasta gamma di campi, in ultima analisi, con l'obiettivo di controllare le malattie zanzare può trasmettere.
Tradizionalmente, controllo arbovirus dipende fortemente l'uso di insetticidi per ridurre le popolazioni di zanzare. Tuttavia, vi è crescente interesse approcci dove le zanzare modificate sono allevate in laboratorio e poi rimesso in popolazioni naturali. Rilasciate le zanzare possono essere modificate geneticamente11,12,13, biologicamente14,15, attraverso irradiazione16, trattamento chimico17,18, o con combinato tecniche19 per sopprimere le popolazioni di zanzare o sostituirli con le zanzare che sono refrattari a arbovirus trasmissione20.
Wolbachia sono batteri che vengono attualmente utilizzati come agente di controllo biologico per gli arbovirus. Diversi ceppi di Wolbachia recentemente sono stati introdotti in Ae. aegypti sperimentalmente mediante microiniezione embrionali21,22,23,24. Questi ceppi riducono la capacità dell'arbovirus di diffondere e replicare nella zanzara, diminuendo la loro trasmissione potenziale23,25,26,27,28 . Wolbachia infezioni vengono trasmessi dalla madre alla prole, tuttavia alcuni ceppi inducono sterilità quando infetti maschi si accoppiano con le femmine non infetti22. Wolbachia-maschi infetti pertanto possono essere rilasciati in grandi quantità per sopprimere popolazioni di zanzara naturale, come recentemente dimostrati in altri15,specie di Aedes 29. Tuttavia, poiché Wolbachia inoltre inibiscono la trasmissione arbovirus in Ae. aegypti, zanzare possono essere rilasciate anche per sostituire le popolazioni native con vettori più poveri. Ae. aegypti infettati sperimentalmente con Wolbachia ora vengono rilasciate nel campo in diversi paesi che utilizzano questo approccio posteriore14,30,31.
Wolbachia-approcci di basati per controllo arbovirus si basano su una solida comprensione delle interazioni tra Wolbachia, la zanzara e l'ambiente. Wolbachia si presentano naturalmente in una vasta gamma di insetti, e le deformazioni introdotte le zanzare sono diverse nei loro effetti32. Come vengono introdotti nuovi tipi di infezione di Wolbachia in Ae. aegypti24, è necessario caratterizzare ogni ceppo per i loro effetti sulla zanzara fitness, riproduzione e arbovirus interferenza in una gamma di condizioni. Rigorosa sperimentazione in laboratorio è pertanto necessaria per valutare il potenziale per i ceppi di Wolbachia avere successo nel campo.
Uscite di campo aperto di Ae. aegypti con Wolbachia infezioni possono spesso richiedono migliaia o decine di migliaia di zanzare per zona di rilascio per essere allevato ogni settimana14,30,31. Il successo delle versioni iniziali può essere migliorato attraverso il rilascio di zanzare di grandi dimensioni per massimizzare la loro fecondità33 e accoppiamento successo34,35. Le zanzare dovrebbero anche essere adattate alle condizioni che essi sperimenteranno nel campo, tuttavia a lungo termine laboratorio allevamento può causare cambiamenti nel comportamento e fisiologia che poteva avere un impatto campo prestazioni36,37, 38.
Descriviamo un semplice protocollo per l'allevamento di Ae. aegypti in laboratorio con attrezzature di base. Questo protocollo è adatto per entrambi selvaggio-tipo e Wolbachia-infettato le zanzare, il posteriore di quale può richiedere particolare attenzione come alcuni ceppi di Wolbachia hanno effetti sostanziali sulla zanzara vita-storia tratti39, 40. le condizioni di allevamento evitare sovraffollamento e competizione per il cibo per produrre le zanzare di una dimensione coerente, che è critica per competenza vettoriale ed esperimenti di fitness e garantisce che le zanzare sono sane per il rilascio di campo41 . Inoltre prendiamo precauzioni per minimizzare laboratorio adattamento e consanguineità riducendo le pressioni selettive e garantire che la prossima generazione è campionata da una grande piscina casuale. Tuttavia, ambienti di laboratorio sono distintamente differenti da condizioni di campo e manutenzione a lungo termine in condizioni rilassate potrebbe ridurre l'idoneità delle zanzare al momento del rilascio in campo37,42,43 . Attraversiamo quindi le femmine da linee di laboratorio ai maschi di campo-raccolti periodicamente, conseguente colonie che sono geneticamente simili per confronti sperimentali e che sono adattati per la destinazione campo popolazione39. I metodi non richiede attrezzature specializzate e possono essere scalati a posteriore decine di migliaia di individui a settimana per i rilasci di campo. Il protocollo inoltre assegna una priorità l'idoneità delle zanzare all'interno e attraverso le generazioni, una considerazione importante per insetti destinati per l'istituzione nelle popolazioni naturali. Il protocollo è adatto per la maggior parte dei laboratori che richiedono manutenzione di Ae. aegypti, particolarmente per i confronti sperimentali dove una qualità costante di zanzare e relatability al campo sono importanti.

<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

Le zanzare Aedes aegypti infettate sperimentalmente con Wolbachia stanno utilizzandi in programmi per controllare la diffusione di arbovirus come dengue, chikungunya e Zika. Wolbachia-zanzare infette possono essere rilasciate nel campo di ridurre dimensioni di popolazione attraverso accoppiamenti incompatibili o trasformare popolazioni con le zanzare che sono refrattari alla trasmissione del virus. Per queste strategie avere successo, le zanzare rilasciate nel campo dal laboratorio devono essere competitive con le zanzare autoctone. Tuttavia, mantenere le zanzare in laboratorio può provocare inbreeding, deriva genetica e adattamento di laboratorio che può ridurre la loro idoneità nel campo e può confondere i risultati degli esperimenti. Per verificare l'idoneità dei diversi Wolbachia infezioni per la distribuzione in campo, è necessario mantenere le zanzare in un ambiente di laboratorio controllato attraverso più generazioni. Descriviamo un protocollo semplice per mantenere le zanzare Ae. aegypti in laboratorio, che è adatto per entrambi Wolbachia-zanzare infette e wild-type. I metodi minimizzare adattamento di laboratorio e implementano esogamici per aumentare la rilevanza degli esperimenti alle zanzare di campo. Inoltre, colonie sono mantenute in condizioni ottimali per massimizzare la loro idoneità per i rilasci di campo aperto.

Figura 4 illustra gli effetti della nutrizione suboptimale sullo sviluppo delle larve di Ae. aegypti . Quando sono disponibili contenitori con 0,25 mg di cibo al larva al giorno o meno, aumenta il tempo di sviluppo per sia i maschi che le femmine, ed è meno sincrona rispetto in contenitori fornita con 0,5 mg di cibo. Se un'alimentazione adeguata non viene fornita per tutta la durata dello sviluppo larvale, questo potrebbe avere un impatto negativo s...

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

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Mantenimento di Aedes aegypti zanzare infettate con Wolbachia

Le zanzare Aedes aegypti infettate con Wolbachia vengono rilasciate in popolazioni naturali di sopprimere la trasmissione dell'arbovirus. Descriviamo i metodi retro Ae. aegypti con Wolbachia infezioni in laboratorio per esperimenti e rilascio di campo, prendendo precauzioni per ridurre al minimo la selezione e l'adattamento di laboratorio.

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 infezione batterica in Drosophila

Ricerca

Biologia

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.

Microiniezione di A. aegypti embrioni per ottenere zanzare transgeniche

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.

Protocollo per la dengue in Zanzare (A. aegypti) e Fenotipo Determinazione infezione

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.

Il mantenimento di Wolbachia in Cell-free medio

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.

Dissezione delle ghiandole salivari e midgut da Ae. aegypti zanzare

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.

Costruire un Mosquito migliore: l&#39;identificazione dei geni Abilitazione malaria e febbre dengue Resistenza in A. gambiae e A. aegypti zanzare

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-mediato Knockdown Gene e<em&gt; In Vivo</em&gt; Saggio La diuresi in femmina adulta<em&gt; Aedes aegypti</em&gt; Le zanzare

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.

Fisiologici Registrazioni e RNA sequenziamento delle appendici gustative del Mosquito febbre gialla<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.

Sistema di coltura di organo del grasso corporeo in Aedes Aegypti, vettore di Virus di 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.

Test migliorato di fecondità e fertilità per Aedes aegypti utilizzando 24 piastre di coltura dei tessuti dei pozzi (piastre EAgaL)

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