3. Establishing crossing between field and laboratory mosquitoes.
Field-caught mosquitoes from Elende were crossed with the FANG An. funestus colony to allow segregation of the resistance allele already at a very high frequency in the population. F1 larvae from the field mosquitoes and larvae from the FANG colony were reared until the pupal stage, then individually placed in 15ml tubes with 2ml of water and stoppered with cotton wool. This was done to obtain virgin female mosquitoes for each strain. Female field-collected mosquitoes were crossed with male FANG to generate a field x FANG line. A reciprocal crossing with the female FANG and the male from the field was also generated. Only the female FANG crossed with males for the field was successfully established. This line was maintained until the F3 generation when bioassays were performed.