Fig. 2: Overexpression validation and phenotypic characterization of candidate genes in transgenic parasites.

a Construction strategy of overexpression transgenic parasites. The purple regions indicate mutation sites. b Fluorescence observation of F₁ generation transgenic parasites. Scale bar, 50 µm. c mCherry expression across different stages of ETH2_0402100^Mut-OE strain. Unsporulated oocyst, sporulated oocyst, sporozoites, schizont, merozoite, and gametocyte. Scale bar, 5 µm. d Indirect immunofluorescence microscopy showing the localization of ETH2_0402100^Mut-OE-mCherry. Mouse anti-Flag mAb was used to detect the Flag-tagged ETH2_0402100^Mut protein followed by FITC-conjugated goat anti-mouse IgG. Scale bar, 10 µm. e Stable expression of the target protein in ETH2_0402100^Mut-OE strain validated by western blot. H was used as the wild-type control. f Oocyst output curves comparing ETH2_0402100^Mut-OE and wild-type H strains. “−” and “+” indicate treatment without and with maduramicin (5 mg/kg), respectively. g Total oocyst output measured during infection. Strain H served as the wild-type control. “−” and “+” indicate treatment without and with maduramicin (5 mg/kg), respectively. Statistical analysis was performed using an unpaired two-tailed Student’s t-test. P-values: ***p < 0.001; ns not significant. Data are presented as mean ± SD. Each dot represents an individual biological replicate (n = 3).