|Home | About | Journals | Submit | Contact Us | Français|
This protocol describes methods for culturing Ae. aegypti. A procedure for egg collection is included and can be used in conjunction with the accompanying fixation, immunohistochemistry, and in situ protocols.
The use of environmentally controlled chambers (insectaries) dedicated to mosquito rearing is encouraged. Adults can be maintained in mesh cages (Fig. 2) located in an insectary (see Christophers, 1960 for a discussion of cage construction). For general rearing, mosquitoes are maintained at 26°C, 84% relative humidity, under a 16 hr light and 8 hr dark cycle with 1 hr crepuscular periods at the beginning and end of each light cycle.
The time in which a mosquito matures to adulthood is dependent upon environmental factors such as temperature, humidity, and nutrition. In the conditions described in this protocol, development proceeds roughly as follows:
After ~3 days, eggs in diapause that have been collected on egg paper (see below) can be used in scheduled rearing.
Protocols for use of animals for blood feeding must be approved by the appropriate Institutional Animal Care and Use Committee. Our protocols typically use anesthetized mice or rats depending on numbers of mosquitoes to be blood fed. For blood feeding, animals should be placed on of the top of the netted cage for ~15 min. Adult female mosquitoes should be aged ~3 days and should be deprived of sucrose solution ~12-24 hr prior to blood feeding.
Egg collections can be made ~3 days following blood feeding, at which time each adult female will lay ~100-150 eggs. The following procedure will allow for collection of eggs that will be used for maintenance of the culture. The procedure differs slightly for timed collections (see V.b) or when collecting eggs for microinjection (see microinjection protocol).
If you wish to obtain embryos synchronized at a particular stage of embryonic development, use a narrower egg laying time (i.e. 30 min.). Following egg collection, place the egg paper onto a layer of wet paper towels, and put the egg paper/paper towels into a sealable sandwich bag or plastic container. Keep the bag in the insectary or in an incubator set at 26° C. Be sure that the egg paper remains damp (but not soaking wet) as you age the embryos to the desired stage.
Problem: Slow development (II. Life cycle); Solution: Overcrowding can slow development, as larvae will not be able to acquire adequate nutrition. Adhere to the cage densities and feeding instructions described throughout this protocol.
Problem: Poor hatching (step III.1); Solution: Do not store eggs more than three months. The time required for eggs to hatch will increase and egg viability will decrease as you approach the three month mark.
Problem: Variable egg-laying (step V); Solutions: For basic colony maintenance, prepare fresh cages every two weeks. Be sure to blood feed the mosquitoes weekly when you need to perform egg collections. Be aware that mosquitoes will lay the majority of their eggs on the first egg paper placed following a blood feed. This makes it difficult to collect eggs in a single cage of mosquitoes more than one time following a single blood meal. To combat this problem, it can be helpful to rear multiple cages of mosquitoes. It is also possible to transfer a small number of blood fed females from a large cage to a smaller egg collection chamber; females remaining in the larger cage can then be used for later egg collections (see Lobo et al., 2006).
The culturing methodology described here allows for fairly routine maintenance of Ae. aegypti. The procedure for egg-laying can be used to collect large numbers of eggs for in situ hybridization analysis of gene expression or immunohistemical analysis of protein expression, both of which are revealing new insight into the development of this vector mosquito (Simanton et al., 2009).
Development of the protocols described here was funded by the following awards NIH/NIAID Award R01 AI 081795, NIH/NINDS Award R15 NS 048904, and an IUSM Research Support Funds Grant to MDS and NIH/NIAID Award RO1 AI 059342 to DWS.
Take a squirt bottle and trim the tip to create a larger opening. Weigh 5 g of liver powder (MP Biomedicals Cat# 900396) and add dH20 to a final volume of 500 ml. Mix and store at 4° C. Shake well before each use.
Combine 9.5 g sucrose with 189 ml distilled water. Mix the reagents and store at 4° C.
Conflicts of interest: none declared