Protocol to determine the effect of temperature on the duration of immature stages of Ae.aegypti

Protocol to determine the effect of temperature on the duration of immature stages of Ae.aegypti
  1. Collect the Aedes eggs using ovitraps
  2. After collecting the ovitraps, keep the ovistrips in the laboratory for 2 -3 days allowing to mature the eggs
  3. Put 01 litre of de-chlorinated water into a enamel tray/s 
  4. Place the egg strips in the trays –  Do not put more than 400 eggs per tray
  5. Add powdered fish food
  6. Allow the larvae to develop its 3rd and 4th stages
  7. Identify the larvae at their 3rd and 4th stages and separate Ae. aegypti and Ae. albopictus
  8. Allow the larvae to develop to pupae
  9. Collect the pupae and place them in a small beaker with de-chlorinated water
  10. Place the beaker containing pupae in a mosquito proof cage for emergence to adults
  11. Leave it for 48 hours for emergence
  12. After emergence keep them for another 3 days and feed them with blood on the 3rd day. If necessary, continue the colony, then feed the adult female mosquitoes with blood at 3-4 days intervals
  13. Separate and transfer  the female mosquitoes to another cage (remove the males)
  14. Keep an egg cup (ovi cup) for laying eggs 48 hours after feeding
  15. Allow the egg cup for laying eggs for 3 days
  16. Keep the egg strips in the laboratory for 2 days allowing to mature the eggs
  17. Count the number of eggs in the strips – can count good and bad eggs separately
  18. Get 6 batches of 50 good eggs from the egg strips
  19. Place each the egg strips in transparent bowls and note the time and date of placing the egg strips into the bowls
  20. Examine the bowls at 8.00am and 4.30 pm daily and  count the number of 1st and 2nd stage larvae, 3rd and 4th instar larvae, pupae, male and female adult mosquitoes
  21. Note the 
    • time/ day of hatching to first instar larvae from the eggs
    • time/day of larval transformation from one larval stage to the next larval stage, to pupae and to male and female adult mosquitoes 
  1. Note the temperature and humidity daily starting from placing the eggs throughout the experiment 
  2. Continue the experiment till all larvae become adults or all the bowls has no larvae
  3. Use the attached format for data recording