In order to determine the CI level, experimental crosses between FF (females) wPipSJ × MM (males) w+PipSJ and FF wPipSJ × MM wPipSJ were performed. Pupae of each line were separated in individual glass tubes to avoid mating once adults emerged. Two days after emergence mass crosses were performed between 25 virgin adults of each sex during two days in a single cage (30 cm × 30 cm). After three days, females were blood-fed for 4 h and twenty-four hours later were removed and individually placed into smaller cages (15 × 15 cm) for oviposition. The eggs raft from each female was placed in containers with 10 mL of dechlorinated water and eggs hatched were quantified under a binocular microscope. Crossing experiments were repeated up to achieve 16 eggs raft per crossing, using five generations of laboratory mosquito lines. The spermatheca of all females that laid eggs was checked to determine the occurrence of spermatozoa and to confirm mating. Infection was detected by using the diagnostic PCR assay.

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