Breeding of mosquitos

Mosquitos produce eggs only after a blood meal. Usually the mosquitos are fed once a week with anaesthesised mice that are placed on a window with mesh of the cage. Two days after feeding eggs can be collected by putting a wet filter paper (best within a small petri disk with water) into the cage. The females will lay eggs on the filter soon after, so that most of the embryos are of the same developmental stage. Eggs can be collected for about two to three days. It is essential to keep the mosquitos at high humidity (90%) at 28¡C, best with a day/night cycle (e. g. 16h day/8h night).

Procedure

  • remove the wet filter paper with the embryos from the cage and transfer them with a fine brush into breeding water. Incubate at 28¡C for 3-4 days
  • feed the larvae with powdered cat food (grind the cat food with a pistle in a mortar). Do use little food.
  • daily exchange the breeding water and clean the tank with water
  • the larvae develop into pupae within 10 to 14 days
  • transfer the pupae with a wide-open pasteur pipette to a separate tank with breeding water. The pupae are not fed.
  • Mosquitos ecclose after ???? days of pupal development. Make to to place the tank timely into a cage.
  • Mosquitos are fed by mineral water and sugar water. Soak a two pieces of cotton wool with water and sugar water. Replace the cotton wools daily

Materials

  • Breeding water:
    1 g/l sea salt (can be obtained in the supermarket) in deinonised water
  • sugar water
    5 g saccharose in 50 ml deionised water, add a little bit of amino benzoic acid tip of a spatula
  • mineral water
    with low carbonic acid level, e. g. Volvic

Fixing mosquito embryos

modified from Goltsev et al, Development 134 (2007) 2415-2424
  • transfer embryos from the wet filter paper with a fine brush to a small net
  • incubate embryos in 20% bleech (Klorix) for 75s to dissolve the chorion
  • wash embryos in water and transfer to a scintillation vial with 5 ml heptan and 4 ml PBS
  • add 1,2 ml of 37% formaldehyd (final 9%) and fix on a shaker for 30 min
  • discard the formaldehyde phase (lower) with a pasteur pipette
  • wash twice with water
  • add 5 ml of water and incubate on a shaker for 30 min
  • remove the aqueous phase
  • add boiling water to the scintillation vial and incubate for 30 s
  • add pieces of ice to quickly cool down and place the vial on ice for 15 min, leave overnight at 4¡C
  • remove the aqueous phase and wash with new heptane
  • add an equal volume of methanol (5 ml)
  • shake vigorously for 1 min
  • incubate for 10-15 min at room temperature, embryos will remain at the interphase
  • transfer embryos to a eppendorf cup
  • wash several times with ethanol
  • store at -20¡C
  • manually remove the endochorion prior to immunhistology with a small canula or fine tungsten needle

Materials:

scintillation vials
PBS
n-heptane
methanol
ethanol
37% formaldehyd
water
microwave
embryo net
klorix