Both male and female adult injection survivors (G0) were mated to the parental wildtype strain (TPRI). Male G0 individuals were crossed to three wild type females, and after 7 days these were pooled into groups of ten males and 30 females. G0 females were mated in pools of ten to approximately 20 wildtype males. Pools were blood fed and after 5 days, eggs were collected as described above. Four ovipositions were collected for all pools and screened under the Leica MZ165FC microscope (Leica Biosystems, Milton-Keynes UK) and images were taken using a Leica DFC camera and the settings: Brightness 82%, saturation 0 and Gamma 0.71 (for white light images) and Brightness 82%, Saturation 0.146 and Gamma 0.40 (mCherry—red fluorescence). Around 20 fluorescent marker positive G1 males and females from the relevant lines were crossed to provide larvae for each viability assay.

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