Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Summary

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.

Abstract

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.

Introduction

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 expand^2,3,4. 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 wit....

Protocol

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 ± 0.5 °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 development^44,45,46

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Wild type Aedes aegypti			Collected from field locations in Queensland, Australia, see Yeap and others39 for details
w Mel-infected Aedes aegypti			Provided by Monash University. Refer to Walker and others23 for information on the strain
w AlbB-infected Aedes aegypti			Provided by Monash University. Refer to Xi and others21 for information on the strain
w MelPop-infected Aedes aegypti			Provided by Monash University. Refer to McMeniman and others22 for information on the strain
Instant dried yeast	Lowan		Stimulates egg hatching. Found in general grocery stores. Other brands may be used
5 L plastic tub	Quadrant	Q110950	Used for hatching and rearing larvae. Other products may be used
Fish Food (Tetramin Tropical Tablets)	Tetra	16152	Provided to larvae as a source of food. Web address: https://www.amazon.com/Tetra-16152-TetraMin-Tropical-10-93-Ounce/dp/B00025Z6SE
Plastic containers			Used for rearing larvae. Any plastic container above 500 mL should be suitable
Glass pipette			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
Clicker counter	RS Pro	710-5212	Used to assist in the counting of larvae, pupae and eggs. Web address: http://au.rs-online.com/web/p/products/7105212/?grossPrice=Y
Rearing trays	Gratnells		Used for rearing larvae. Web address: http://www.gratnells.com
Nylon mesh			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
Cages	BugDorm	DP1000	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
35 mL plastic cup	Huhtamaki	AA272225	Used to provide water or sucrose to adult mosquitoes. Other brands may be used
35 mL plastic cup lid	Huhtamaki	GB030005	Used to provide sucrose to adult mosquitoes. Other brands may be used
Cotton wool	Cutisoft	71841-13	Moist cotton wool is provided as a source of water to adults. Other brands may be used
White Sugar			Provided as a source of sugar to adult mosquitoes. Found in general grocery stores
Rope	M Recht Accessories	C323C/W	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/
Plastic cup (large)			Used as an oviposition container. Any plastic cup that holds 100 mL of water should be suitable
Sandpaper	Norton Master Painters	CE015962	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
Filter paper	Whatman	1001-150	Used as an alternative oviposition substrate. Other brands may be used
Latex gloves	SemperGuard	Z560979	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/