We acknowledge Heng Lin Yeap, Chris Paton, Petrina Johnson and Clare Doig for their contributions to the development of our colony maintenance methods, and three anonymous reviewers for their suggestions which helped to improve the manuscript. Our research is supported by a program grant and fellowship to AAH from the National Health and Medical Research Council and a translation grant from the Wellcome Trust. PAR is a recipient of an Australian Government Research Training Program Scholarship.

Blood feeding of mosquitoes on human subjects was approved by the University of Melbourne Human Ethics Committee (approval #: 0723847). All volunteers provided informed written consent.
1. Larval Rearing
NOTE: Mosquitoes are held at 26 &#177; 0.5 &#176;C and 50-70% relative humidity, with a 12:12 h (light:dark) photoperiod for this colony maintenance protocol. These conditions are similar to the average climatic conditions in Cairns, Australia and within the optimal thermal range for Ae. aegypti survival and development44,45,46. High temperatures can result in the loss of Wolbachia infections from mosquito colonies and should be avoided47. We maintain at least 500 individuals per population to minimize inbreeding; maintaining colonies of a smaller size can have fitness consequences [Ross et al. unpublished]. Under these conditions and assuming adequate nutrition, the average generation time is 28 days (see Table 1).
<ol>
	<li>Submerge the eggs on substrate in trays (Figure 1A) containing 3 L of water (reverse osmosis water or aged tap water, generated by leaving tap water in trays for 24 h prior to use), ~300 mg of fish food (one crushed tablet, see Table of Materials) and a few grains of active dry yeast to induce hatching48.</li>
	<li>One day after hatching, use a glass pipette to transfer approximately 500 larvae to trays containing 4 L of water (Figure 1B), count using a clicker counter. Add two crushed fish food tablets to each tray. If needed, use containers of different sizes for rearing larvae (Figure 1A), but keep larval densities below 0.5 larvae/mL to avoid overcrowding.</li>
	<li>Check the trays daily to ensure that the larvae have sufficient food; add approximately two food tablets to the trays every two days. Provide food ad libitum but ensure that 0.5 mg/larva/day is available&#160;during this period to ensure development is synchronous and body size is consistent, otherwise the results of experiments may be confounded (see Representative Results).</li>
	<li>Take care to avoid the overfeeding of larvae, particularly in smaller rearing containers with less water surface area and volume. If the water looks cloudy or if there is significant larval mortality, replace it with fresh water; mortality should be negligible if larvae are fed optimally.</li>
</ol>
2. Adult Emergence
NOTE: Larvae will begin to pupate from five days after hatching if well fed and the majority should pupate by seven days after hatching. Adults will begin emerging approximately two days after pupation if maintained optimally at 26 &#176;C (see Representative Results). Larval development is typically unaffected by Wolbachia infections when ample food is provided23,39,49.
<ol>
	<li>Seven days after hatching pour the entire contents of the tray through a fine mesh (pore size 0.4 mm). Keep the filtered larval water for later use in ovicups (see &#34;Blood Feeding and Oviposition&#34; section). Invert the mesh and dip it into a plastic container with 200 mL of water to transfer the pupae. Provide additional food if any larvae remain.</li>
	<li>Prepare the adult emergence cages (Figure 1C) by providing two cups of 10% sucrose solution (Figure 1F) and two cups of moist cotton&#160;wool to prevent desiccation (Figure 1E).</li>
	<li>If the pupae do not need to be sorted by sex, place the lidded containers of pupae into the cage and leave the lid slightly ajar to allow the adults to emerge into the cage. Alternatively, place an inverted funnel over the container to minimize drowning. Ensure all adults have emerged before removing the container from the cage to prevent selecting against slow developers.</li>
</ol>
3. Pupal Sexing for Outcrossing
<ol>
	<li>If the pupae do need to be sorted by sex (e.g., for outcrossing), pipette the pupae from larval trays and separate the sexes (Figure 2) into plastic containers (Figure 1A) with 200 mL of water every 24 h until the desired number of each sex has been reached. Place lids on the containers and leave them closed.</li>
	<li>Adults will emerge into the containers; confirm their sex&#160;before releasing into cages (Figure 2C). Remove any adults sexed incorrectly with an aspirator within 24 h of emergence before they reach sexual maturity. Once the sexes have been confirmed, release the adults into cages every 24 h.</li>
	<li>To obtain Wolbachia-infected colonies of a similar genetic background to a natural population, outcross by adding Wolbachia-infected females from laboratory colonies to cages of uninfected males derived from eggs collected by ovitraps in the field39, maintaining the prescribed density of 500 individuals per population.
	<ol>
		<li>Repeat the outcrossing for at least three consecutive generations to produce colonies that are at least 87.5% similar genetically to the field population39. Critical: Ensure that the sexes are correct at this stage (see step 3.1).</li>
	</ol>
	</li>
	<li>Female Ae. aegypti are usually refractory to further insemination within hours of mating50. When outcrossing colonies, allow the females and males to mature in separate cages for two days and then aspirate the females into the male cage to provide an equal opportunity to all males.</li>
</ol>
4. Blood Feeding and Oviposition
<ol>
	<li>Wait for at least three days after the last female has emerged before blood feeding to allow ample time to mature. Blood feed the females within two weeks of emergence to prevent excessive mortality, particularly for mosquitoes with Wolbachia infections that adversely affect longevity22,24,49. Remove the sugar cups the day prior to feeding to improve feeding rates.
	<ol>
		<li>Ask a volunteer to insert their forearm into the cage to allow the female mosquitoes to feed. Most females should feed to repletion within 5 min, but to reduce selection against slow feeders, leave the forearm in the cage for 15 min, or until all females are visibly engorged; a latex glove to protect the hand from bites is optional but recommended.</li>
		<li>Two days after blood feeding, place two plastic cups containing larval rearing water and lined with a strip of sandpaper (Figure 1G) (or filter paper (Figure 1H)) into the cage for females to lay eggs. Partially submerge the sandpaper strip in the water to keep it moist. Remove other sources of water to prevent the females from laying their eggs outside the oviposition cup. 
		NOTE: Tap water may be used in the cups, but larval-rearing water encourages oviposition51,52 and females will lay their eggs more synchronously.</li>
	</ol>
	</li>
</ol>
5. Egg Collection and Conditioning
<ol>
	<li>The females will lay eggs on the sandpaper just above the water line; collect and replace the strips of sandpaper daily until no more eggs are laid. Note that oviposition may continue for up to one week.</li>
	<li>Partially dry the sandpaper strips by gently blotting them on a paper towel for 30 s, taking care not to dislodge the eggs. Then, wrap the strips in a sheet of dry paper towel and place it into a sealable plastic bag (Figure 1I).&#160;</li>
	<li>Check the condition of the eggs under a dissecting microscope (Figure 3). If the sandpaper strips are too wet, eggs may hatch before being submerged in water (Figure 3B), but if dried too harshly, eggs may collapse (Figure 3C).</li>
	<li>The eggs can be hatched at any time beyond three days post-collection; hatch all eggs from each colony, collected across all days, in the same container of water to ensure that the next generation is sampled from a large, random pool of individuals.</li>
	<li>For long-term storage, keep the eggs in a sealed container at a high (&#62; 80%) humidity at around 20 &#176;C. Under these conditions, eggs without Wolbachia can be stored&#160;for several months while maintaining high hatch rates53,54.</li>
	<li>As some Wolbachia infections greatly reduce the viability of eggs with age49,55, hatch the eggs from Wolbachia-infected lines within one week of collection to prevent excessive mortality for the relevant strains. Blood feed the females again after one week if more eggs are needed.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Day</td>
			<td colspan="3">Step</td>
		</tr>
		<tr>
			<td>0</td>
			<td colspan="3">Hatch eggs</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="3">Count larvae into trays</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="3">Transfer larvae and pupae to colony cages</td>
		</tr>
		<tr>
			<td>17</td>
			<td colspan="3">Blood feed female adults</td>
		</tr>
		<tr>
			<td>21</td>
			<td colspan="3">Begin collecting eggs</td>
		</tr>
		<tr>
			<td>25</td>
			<td colspan="3">Finish collecting eggs</td>
		</tr>
		<tr>
			<td>28</td>
			<td colspan="3">Hatch eggs</td>
		</tr>
	</tbody>
</table>
Table 1: Overview of the Ae. aegypti colony maintenance schedule at 26 &#176;C. The timing of the female blood-feeding and the hatching of eggs is flexible, but long durations at these stages should be avoided, particularly for mosquitoes infected with Wolbachia, in order to minimize mortality. Following this schedule minimizes selection against mosquitoes that are fast or slow to develop or mature at all life stages, provided that larvae are fed optimally.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56124/56124fig1.jpg" /> 
Figure 1: Equipment used for rearing Ae. aegypti in the laboratory. (A) Plastic containers used for hatching eggs or rearing larvae with volumes of 500, 750 and 5,000 mL (from left to right). (B) Trays used for rearing larvae at a controlled density, usually 500 larvae in 4 L of water. (C) 19.7 L and (D) 3 L cages used for housing adults. A density of 25 adults or less per liter should be maintained to provide sufficient space. (E) 35 mL cup with moist cotton wool provided as a source of water to adults. (F) 35 mL cup with sucrose solution provided through a cord or dental wick as a source of sugar. (G-H) Cups filled with larval rearing water and lined with an oviposition substrate of sandpaper or filter paper (G and H, respectively). (I) Zip-lock bag used for storage of sandpaper strips or filter paper. Black spots on sandpaper are mosquito eggs. <a href="//cloudfront.jove.com/files/ftp_upload/56124/56124fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56124/56124fig2.jpg" /> 
Figure 2: Lateral (A) and dorsal (B) views of pupae and adult Ae. aegypti (C) demonstrating their sexual dimorphism. Males are positioned on the left and females on the right in each panel. When optimally fed, male and female pupae are distinguished by size; females are larger than males (A) and have a relatively bulbous cephalothorax compared to males which have flatter sides (B). Male adults are easily distinguished from females under all rearing conditions, primarily by their plumose antennae and long palps. <a href="//cloudfront.jove.com/files/ftp_upload/56124/56124fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56124/56124fig3.jpg" /> 
Figure 3. Four day old Ae. aegypti eggs under different conditions. (A) Intact eggs on sandpaper strips maintained at a high (&#62; 80%) humidity but without any visible moisture. Hatch rates should be above 90% for wild-type Ae. aegypti if maintained correctly. (B) Eggs that hatch before being submerged in water (precocious hatching) are distinguished by a detached egg cap and visible larva. This indicates that the sandpaper strip was kept too moist. (C) Eggs that are dried too harshly may collapse, and are clearly visible by their concave appearance. If the sandpaper becomes stiff this also indicates that the eggs may be too dry. <a href="//cloudfront.jove.com/files/ftp_upload/56124/56124fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The authors declare that they have no competing financial interests.

Following the protocol presented here for the maintenance of Wolbachia-infected Ae. aegypti should ensure that healthy mosquitoes of a consistent quality are produced for experiments and open field releases. In contrast to other protocols that prioritize the production of mass quantities of mosquitoes (see reference57), the methods are focused on maximizing their fitness, both within generations by implementing relaxed rearing conditions, and across generations by minimizing inbr...

Aedes aegypti mosquitoes are responsible for transmitting some of the most important arboviruses in the world, including dengue, Zika and chikungunya1. These viruses are becoming an increasing threat to global health as the widespread distribution of Ae. aegypti in the tropics continues to expand2,3,4. Female Ae. aegypti preferentially feed on human blood5 and thus tend to live in close proximity to humans, particularly in urban areas where populations are most dense. Through this close association with humans they have also adapted to breed in artificial habitats, including tires, pots, gutters and water tanks6,7. Ae. aegypti also readily adapt to laboratory environments where they can be maintained without any special requirements after being collected directly from the field, unlike some other species in the Aedes genus8,9,10. Their ease of maintenance has seen them studied widely in the laboratory in a broad range of fields, ultimately aiming to control the diseases mosquitoes may transmit.
Traditionally, arboviral control relies heavily on the use of insecticides to reduce mosquito populations. However, there is increasing interest in approaches where modified mosquitoes are reared in the laboratory and then released into natural populations. Released mosquitoes may be modified genetically11,12,13, biologically14,15, through irradiation16, chemical treatment17,18, or with combined techniques19 to either suppress populations of mosquitoes or replace them with mosquitoes that are refractory to arboviral transmission20.
Wolbachia are bacteria that are currently being used as a biological control agent for arboviruses. Several strains of Wolbachia were recently introduced into Ae. aegypti experimentally using embryonic microinjection21,22,23,24. These strains reduce the capacity of arboviruses to disseminate and replicate in the mosquito, diminishing their transmission potential23,25,26,27,28. Wolbachia infections are transmitted from mother to offspring, however certain strains induce sterility when infected males mate with uninfected females22. Wolbachia-infected males can therefore be released in large quantities to suppress natural mosquito populations, as recently demonstrated in other Aedes species15,29. However, since Wolbachia also inhibit arboviral transmission in Ae. aegypti, mosquitoes can also be released to replace native populations with poorer vectors. Ae. aegypti infected experimentally with Wolbachia are now being released into the field in several countries using this latter approach14,30,31.
Wolbachia-based approaches for arboviral control rely on a sound understanding of the interactions between Wolbachia, the mosquito and the environment. Wolbachia occur naturally in a broad range of insects, and the strains introduced into mosquitoes are diverse in their effects32. As new Wolbachia infection types are introduced into Ae. aegypti24, it is necessary to characterize each strain for their effects on mosquito fitness, reproduction and arboviral interference under a range of conditions. Rigorous experimentation in the laboratory is therefore required to evaluate the potential for Wolbachia strains to succeed in the field.
Open field releases of Ae. aegypti with Wolbachia infections can often require thousands to tens of thousands of mosquitoes per release zone to be reared each week14,30,31. The success of initial releases can be improved by releasing mosquitoes of a large size to maximize their fecundity33 and mating success34,35. Mosquitoes should also be adapted to the conditions they will experience in the field, however long-term laboratory rearing may cause changes in behavior and physiology which could impact field performance36,37,38.
We describe a simple protocol for rearing Ae. aegypti in the laboratory using basic equipment. This protocol is suitable for both wild-type and Wolbachia-infected mosquitoes, the latter of which can require special attention as some Wolbachia strains have substantial effects on mosquito life-history traits39,40. The rearing conditions avoid overcrowding and competition for food to produce mosquitoes of a consistent size, which is critical for vector competence and fitness experiments, and ensures that the mosquitoes are healthy for field release41. We also take precautions to minimize laboratory adaptation and inbreeding by reducing selective pressures and ensuring that the next generation is sampled from a large, random pool. However, laboratory environments are distinctly different from field conditions, and long-term maintenance under relaxed conditions could reduce the fitness of mosquitoes upon release into the field37,42,43. We therefore cross females from laboratory lines to field-collected males periodically, resulting in colonies that are genetically similar for experimental comparisons and that are adapted to the target field population39. The methods do not require any specialized equipment and can be scaled up to rear tens of thousands of individuals per week for field releases. The protocol also prioritizes the fitness of mosquitoes within and across generations, an important consideration for insects destined for establishment in natural populations. The protocol is suitable for most laboratories that require maintenance of Ae. aegypti, particularly for experimental comparisons where a consistent quality of mosquitoes and relatability to the field are important.

<table><tbody><tr><td>Wild type &lt;em&gt;Aedes aegypti&lt;/em&gt;</td><td></td><td></td><td>Collected from field locations in Queensland, Australia, see Yeap and others&lt;sup class=&quot;xref&quot;&gt;39&lt;/sup&gt; for details</td></tr><tr><td>&lt;em&gt;w&lt;/em&gt; Mel-infected &lt;em&gt;Aedes aegypti&lt;/em&gt;</td><td></td><td></td><td>Provided by Monash University. Refer to Walker and others&lt;sup class=&quot;xref&quot;&gt;23&lt;/sup&gt; for information on the strain</td></tr><tr><td>&lt;em&gt;w&lt;/em&gt; AlbB-infected &lt;em&gt;Aedes aegypti&lt;/em&gt;</td><td></td><td></td><td>Provided by Monash University. Refer to Xi and others&lt;sup class=&quot;xref&quot;&gt;21&lt;/sup&gt; for information on the strain</td></tr><tr><td>&lt;em&gt;w&lt;/em&gt; MelPop-infected &lt;em&gt;Aedes aegypti&lt;/em&gt;</td><td></td><td></td><td>Provided by Monash University. Refer to McMeniman and others&lt;sup class=&quot;xref&quot;&gt;22&lt;/sup&gt; for information on the strain</td></tr><tr><td>Instant dried yeast</td><td>Lowan</td><td></td><td>Stimulates egg hatching. Found in general grocery stores. Other brands may be used</td></tr><tr><td>5 L plastic tub</td><td>Quadrant</td><td>Q110950</td><td>Used for hatching and rearing larvae. Other products may be used</td></tr><tr><td>Fish Food (Tetramin Tropical Tablets)</td><td>Tetra</td><td>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><td>Plastic containers</td><td></td><td></td><td>Used for rearing larvae. Any plastic container above 500 mL should be suitable</td></tr><tr><td>Glass pipette</td><td></td><td></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><td>Clicker counter</td><td>RS Pro</td><td>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><td>Rearing trays</td><td>Gratnells</td><td></td><td>Used for rearing larvae. Web address: http://www.gratnells.com</td></tr><tr><td>Nylon mesh</td><td></td><td></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><td>Cages</td><td>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><td>35 mL plastic cup</td><td>Huhtamaki</td><td>AA272225</td><td>Used to provide water or sucrose to adult mosquitoes. Other brands may be used</td></tr><tr><td>35 mL plastic cup lid</td><td>Huhtamaki</td><td>GB030005</td><td>Used to provide sucrose to adult mosquitoes. Other brands may be used</td></tr><tr><td>Cotton wool</td><td>Cutisoft</td><td>71841-13</td><td>Moist cotton wool is provided as a source of water to adults. Other brands may be used</td></tr><tr><td>White Sugar</td><td></td><td></td><td>Provided as a source of sugar to adult mosquitoes. Found in general grocery stores</td></tr><tr><td>Rope</td><td>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><td>Plastic cup (large)</td><td></td><td></td><td>Used as an oviposition container. Any plastic cup that holds 100 mL of water should be suitable</td></tr><tr><td>Sandpaper</td><td>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><td>Filter paper</td><td>Whatman</td><td>1001-150</td><td>Used as an alternative oviposition substrate. Other brands may be used</td></tr><tr><td>Latex gloves</td><td>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 mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Figure 4 demonstrates the effects of suboptimal nutrition on the development of Ae. aegypti larvae. When containers are provided with 0.25 mg of food per larva per day or less, the development time increases for both males and females, and is less synchronous than in containers provided with 0.5 mg of food. If adequate food is not provided throughout the duration of larval development, this could have an adverse impact on the maintenance schedule. Sl...

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

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

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

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

Research

JoVE Journal

Biology

14.1K Views.  Bio21 Institute and University of Melbourne. Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Video: Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL&#160;(Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL&#160;strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL&#160;program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program&#160;there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL&#160;control program,&#160;released as either pupae or adults11,12.
To suppress a mosquito population using RIDL&#160;a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL&#160;strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL&#160;males for a control program.

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

To achieve population suppression of Aedes aegypti using the RIDL&#174; (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the ...

Environment

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.

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

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

Genetics

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.

The protocols reported here illustrate three alternative ways to assess the performance of genetically-engineered mosquitoes destined for vector control in laboratory-contained small cage trials. Each protocol is tailored to the specific modification the mosquito strain bears (gene drive or non-gene drive) and the types of parameters measured.

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control ...

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

Four methods were used to detect intracellular Wolbachia, which complemented each other and improved the detection accuracy of Wolbachia infection of Aedes albopictus-derived Aa23 and Aa23-T&#160;cured of native Wolbachia infection using antibiotics.

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation ...

Immunology and Infection

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

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.

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

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito.

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no ...

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain&#8217;s fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.&#160;
Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.&#160;

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

Here, we present a protocol for studying mating competitiveness of male Aedes aegypti using fluorescent dye as a marker. Female mosquitoes are exposed to both marked and unmarked males for copulation. Post mating, their spermathecae are examined under a fluorescence microscope to determine their mating partner.

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for ...

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

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Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes