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

Vi erkänner Heng Lin Yeap, Chris Paton, Petrina Johnson och Clare Doig för deras bidrag till utvecklingen av vår koloni underhåll metoder och tre anonyma recensioner för deras förslag som bidrog till att förbättra manuskriptet. Vår forskning stöds av ett program grant och gemenskap till AAH från National Health och medicinska forskningsrådet och en översättning bevilja från Wellcome Trust. PAR är en mottagare av en australiensisk regering forskning utbildning Program stipendium.

Blod utfodring av myggor på människor och godkändes av University of Melbourne mänskliga etikkommittén (godkännande nr: 0723847). Alla volontärer som informerade skriftliga samtycke.
1. larval uppfödning
Obs: Myggor hålls på 26 ± 0,5 ° C och 50-70% relativ luftfuktighet, med en 12:12 h (ljus: mörker) fotoperiod för denna koloni underhållsprotokoll. Dessa villkor är liknande till genomsnittliga klimatförhållandena i Cairns, Australien och inom optimal...

Efter protokollet presenteras här för underhåll av Wolbachia-infekterade Ae. aegypti bör säkerställa att friska myggor av en konsekvent kvalitet produceras för experiment och öppna fältet utgåvor. I motsats till andra protokoll som prioriterar produktionen av massa mängder mygg (se referens57), är metoderna inriktade på att maximera deras fitness, både inom generationer av avslappnad uppfödning genomförandevillkor, och över generationer genom att minimera inavel,...

Aedes aegypti myggor är ansvarig för att överföra några av de viktigaste arboviruses i världen, inklusive Zika, denguefeber och chikungunya1. Dessa virus blir ett allt större hot mot global hälsa eftersom de omfattande distributionen av Ae. aegypti i tropikerna fortsätter att expandera2,3,4. Kvinnliga Ae. aegypti livnär prioriterat sig på människoblod5 och därmed tenderar att leva i nära närhet till människor, särskilt i stadsområden där populationer är mest tät. Genom detta nära samarbete med människor har de också anpassade för att häcka i konstgjorda livsmiljöer, inklusive däck, krukor, rännor och vatten tankar6,7. AE. aegypti också lätt anpassa sig till laboratoriemiljöer där de kan upprätthållas utan särskilda krav efter samlas in direkt från fältet, till skillnad från vissa andra arter i Aedes släkte8, 9,10. Deras enkelt underhåll har sett dem studerade allmänt i laboratorium i ett brett spektrum av områden, i slutändan syftar till att kontrollera sjukdomar myggor kan överföra.
Traditionellt har beroende arboviral kontroll starkt av användning av insekticider att minska mosquito populationer. Dock finns det ökande intresse för metoder där modifierade myggor är uppfödda i laboratoriet och sedan släpps ut i naturliga populationer. Släppta myggor kan modifieras genetiskt11,12,13, biologiskt14,15, genom bestrålning16, kemisk behandling17,18, eller med kombinerade tekniker19 för att undertrycka populationer av myggor eller ersätta dem med myggor som är refraktära mot arboviral överföring20.
Wolbachia är bakterier som används för närvarande som en biologisk kontroll agent för arboviruses. Flera stammar av Wolbachia infördes nyligen i Ae. aegypti experimentellt med hjälp av embryonala Mikroskop21,22,23,24. Dessa stammar minska kapaciteten för arboviruses att sprida och replikera i mygga, minskar deras överföring potentiella23,25,26,27,28 . Wolbachia infektioner överförs från mor till avkomman, men vissa stammar inducera sterilitet när infekterade hanar parar sig med icke-infekterade kvinnor22. Wolbachia-infekterade män kan därför släppas i stora mängder för att undertrycka naturliga mygga populationer, som nyligen visat i andra Aedes arter15,29. Dock eftersom Wolbachia också hämma arboviral överföring i Ae. aegypti, kan myggor också frigöras för att ersätta inhemska bestånd med fattigare vektorer. Ae. aegypti experimentellt infekterade med Wolbachia släpps nu i fältet i flera länder använder denna senare metoden14,30,31.
Wolbachia-baserade strategier för arboviral kontroll är beroende av en god förståelse av samspelet mellan Wolbachia, mygga och miljö. Wolbachia förekommer naturligt i en rad insekter och de stammar som införs i myggor är olika i deras effekter32. Eftersom nya Wolbachia infektion typer förs in Ae. aegypti24, är det nödvändigt att karakterisera varje stam för sina effekter på mosquito fitness, reproduktion och arboviral störningar under en rad villkor. Rigorösa experiment i laboratoriet är därför nödvändigt att utvärdera potentialen för Wolbachia stammar att lyckas i fältet.
Öppna fältet utgåvor av Ae. aegypti med Wolbachia infektioner kan ofta kräva tusentals till tiotusentals myggor per utgåva zon vara uppfödda varje vecka14,30,31. Framgången för första utgåvor kan förbättras genom att släppa myggor av en stor storlek för att maximera sin fruktsamhet33 och parning framgång34,35. Myggor bör också anpassas till de förhållanden som de kommer att uppleva i fältet, men långsiktigt laboratorium uppfödning kan orsaka förändringar i beteende och fysiologi som kan påverka området prestanda36,37, 38.
Vi beskriver ett enkelt protokoll för uppfödning Ae. aegypti i laboratorium med hjälp av grundläggande utrustning. Detta protokoll är lämplig för både vildtyp och Wolbachia-infekterade myggor, den senare som kan kräva särskild uppmärksamhet eftersom vissa Wolbachia stammar har betydande effekter på mosquito livshistoria drag39, 40. uppfödning villkoren undvika överbefolkning och konkurrens om mat att producera myggor av konsekvent storlek, vilket är avgörande för vektor kompetens och lämplighet experiment, och säkerställer att myggen är friska för fältet övergång41 . Vi också vidta försiktighetsåtgärder för att minimera laboratorium anpassning och inavel genom att minska selektiva påtryckningar och att se till att nästa generation samplas från en stor, slumpmässiga pool. Men laboratoriemiljöer skiljer sig tydligt från fältförhållanden och långsiktigt underhåll under avslappnade förhållanden kunde minska av myggor vid utsättning i fält37,42,43 lämplighet . Vi därför passera honor från laboratoriet linjer till fältet-samlat män regelbundet, vilket resulterar i kolonier som är genetiskt lika för experimentell jämförelser och som är anpassade till de mål fältet befolkningen39. Metoderna kräver inte någon specialiserad utrustning och kan skalas upp till bakre tiotals tusentals individer per vecka för fältet utgåvor. Protokollet prioriterar också lämplighet myggor inom och mellan generationer, en viktig faktor för insekter som är avsedda för etablering i naturliga populationer. Protokollet är lämplig för de flesta laboratorier som kräver underhåll av Ae. aegypti, särskilt för experimentell jämförelser där en konsekvent kvalitet av myggor och relatability till fältet är viktiga.

<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

Aedes aegypti myggor experimentellt infekterade med Wolbachia utnyttjas i program för att kontrollera spridningen av arboviruses såsom Zika, denguefeber och chikungunya. Wolbachia-infekterade myggor kan släppas ut i fältet antingen minska befolkningen storlekar genom oförenliga parningar eller att omvandla populationer med myggor som är refraktära mot virus överföring. För dessa strategier för att lyckas, måste myggen släpps ut i fältet från laboratoriet vara konkurrenskraftiga med infödda myggor. Dock kan att upprätthålla myggor i laboratoriet resultera i inavel, genetisk drift och laboratorium anpassning som kan minska deras lämplighet i fältet och kan blanda ihop resultaten av experiment. För att testa lämpligheten av olika Wolbachia infektioner för distribution i fältet, är det nödvändigt att bibehålla myggor i kontrollerad laboratoriemiljö över flera generationer. Vi beskriver ett enkelt protokoll för att upprätthålla Ae. aegypti myggor i laboratoriet, som är lämplig för både Wolbachia-infekterade och vildtyp myggor. Metoderna minimera laboratorium anpassning och genomföra korsningar för att öka relevansen av experiment fältet mygg. Dessutom bibehålls kolonier under optimala förhållanden att maximera deras lämplighet för öppna fältet utgåvor.

Figur 4 visar effekterna av suboptimal kost på utvecklingen av Ae. aegypti larver. När behållarna är försedda med 0,25 mg mat per larv per dag eller mindre, utvecklingstiden ökar för både män och kvinnor, och är mindre synkron än i behållare försedd med 0,5 mg av mat. Om tillräcklig föda inte tillhandahålls under hela larval development, kan detta ha en negativ inverkan på underhållsschema. Långsam-utveckla individer löper risk at...

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

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Att upprätthålla Aedes aegypti myggor infekterade med Wolbachia

Aedes aegypti myggor infekterade med Wolbachia släpps in i naturliga populationer att dämpa överföringen av arboviruses. Vi beskriva metoder till bakre Ae. aegypti med Wolbachia infektioner i laboratorium för experiment och fältet release, vidta försiktighetsåtgärder för att minimera laboratorium anpassning och urval.

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 bakterieinfektion i Drosophila

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.

Mikroinjektion av A. aegypti Embryon Skaffa Transgena myggor

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.

Protokoll för Dengue Infektioner i Myggor (A. aegypti) och infektion Fenotyp Fastställande

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.

Att upprätthålla Wolbachia i Cell-fri Medium

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.

Dissekering av midgut och spottkörtlar från AE. aegypti Myggor

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.

Bygga en bättre Mosquito: Identifiera gener Aktivera malaria och denguefeber Motståndet i A. gambiae och A. aegypti myggor

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-mediated gene Knockdown och<em&gt; In Vivo</em&gt; Diures Analys hos vuxna kvinnliga<em&gt; Aedes aegypti</em&gt; Myggor

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.

Fysiologiska Inspelningar och RNA sekvensering av de smak bihang till Gula-febern Mosquito<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.

Fett kroppen kultur organsystem i Aedes Aegypti, en vektor av zikaviruset

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.

Förbättrad fecundity och fertilitetsanalys för Aedes aegypti med hjälp av 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 ...