Fig. 4: Conditional knockdown of PbARK2 identifies its essential role in oocyst development and sporogony.
A The number of exflagellation centres per field of Pclag-ark2 (black bar) compared with WT-GFP (white bar) parasites at the end of male gametogony. Shown is mean ± SD; n = 3 independent experiments. B Percentage ookinete conversion for Pclagark2 (black bar) and WT-GFP (white bar) parasites. Ookinetes were identified by reactivity with 13.1 antibody and successful differentiation into elongated ‘banana-shaped’ ookinetes. Shown is mean ± SD; n = 3 independent experiments. Scale bar = 5 μm. C Total number of GFP-positive oocysts per infected mosquito in Pclagark2 (black bar) and WT-GFP (white bar) parasites at 7, 14 and 21-day post infection (dpi). Shown is mean ± SD; n = 3 independent experiments (with >15 mosquitoes for each). Multiple comparison t test (non-parametric) showed significant differences in oocyst number. ***P < 0.001; ns non-significant. (P values: 0.000145—Day 7; 0.053163—Day 10; 0.000010—Day 14; 0.000193—Day 21). D Midguts at ×10- and ×63 magnification showing fluorescent oocysts of Pclagark2 and WT-GFP lines at 7, 14 and 21 dpi. More than 20 images were analysed in more than three different experiments. Scale bar = 50 μm (×10) or 20 μm (×63). E Oocyst sizes of Pclagark2 and WT-GFP lines at 7, 14 and 21 dpi. n = 3 independent experiments (with >10 mosquitoes for each and minimum 30 oocysts counted. Multiple comparisons t test, with post hoc test of Holm–Sidak showed significant differences in relative expression. **P < 0.01, ***P < 0.001. The exact P values and other statistics detail can be found in source data file. F Total sporozoite number in salivary glands of Pclagark2 (black bar, not visible) and WT-GFP (white bar) parasites, showing mean ± SD; n = 3 independent experiments. G Rescue experiment showing male-derived allele of Pclagark2 confers defect and is complemented by ‘female’ Δdozi parasite line. Shown is mean ± SD; n = 3 independent experiments. H RNA-seq analysis showing upregulated and downregulated genes in Pclag-ark2 parasites compared to WT-GFP parasites. I Expression level validation of relevant selected genes from the RNA-seq data using qRT-PCR. Shown is mean ± SEM; n = 3 independent experiments. Multiple comparisons t test, with post hoc test of Holm–Sidak showed significant differences in relative expression. **P < 0.01, ***P < 0.001. The exact P values and other statistics detail can be found in source data file. Source data are provided as a Source Data file.
