Fig. 3. NF54/iGP1_D8 and NF54/iGP2_E9 gametocytes complete their life cycle in the mosquito vector and produce infectious sporozoites.

a NF54/iGP1_D8 (orange), NF54/iGP2_E9 (blue), and NF54 wt control stage V gametocytes (green) were fed to female Anopheles stephensi mosquitoes on day 10, 13, and 14 of gametocytogenesis in two independent SMFA experiments. The violin plots show the distribution of the number of oocysts detected in each of the 20 mosquitoes dissected per feed, with open circles and triangles representing data from SMFA replicates 1 and 2, respectively (left y axis). The median (thick red line) and upper and lower quartiles (thin red lines) are indicated. Closed red circles represent the mean oocyst prevalence (number of infected mosquitoes) determined for each of the two replicate feeds (right y axis). NF54 wt day 10 and day 13 gametocytes were only included in SMFA replicate 1, and for SMFA replicate 2, only 10 mosquitoes infected with NF54 wt day 14 gametocytes have been dissected. b Mean number of salivary gland sporozoites per oocyst (left y axis) and per mosquito (closed red circles; right y axis) 17 days after infection with NF54/iGP1_D8 (orange), NF54/iGP2_E9 (blue), and NF54 wt control day 14 gametocytes (green) (SMFA replicate 2 data). Values represent the results from a single experiment (≥26 mosquitoes dissected per infected batch). c Confocal microscopy IFA images showing intracellular parasites after infection of primary human hepatocytes with NF54/iGP1_D8, NF54/iGP2_E9, and NF54 wt control sporozoites. Parasites were stained with α-PfHSP70 (cytosol; red) and α-PfEXP2 antibodies (parasitophorous vacuolar membrane; purple). α-GFP antibodies were used to test for potential ectopic expression of GDV1 in liver stages. Nuclei were stained with DAPI. Images are representative of a single experiment. Scale bar, 18 µm.