NcPIS_3 <i>Plasmodium</i> ookinetes fail to produce normal oocysts.

<p>(A) Oocyst formation of NcPI-S expressing clones (<i>NcPIS_3</i> and <i>NcPIS_1</i>) compared to that of the parental WT strains (<i>GFPp</i> and <i>ANKA</i> respectively) and of <i>NcPISmut</i> compared to <i>NcPIS_3</i> 7–10 days post infection. Four sets of paired infection experiments were included in the case of <i>NcPIS_3</i>/<i>GFPp</i>, two in the case of <i>NcPISmut</i>/<i>NcPIS_3</i> and three in the case of <i>NcPIS_1</i>/ANKA 2.34 paired infections. Error bars; SEM; P<0.001, Mann-Whitney test, in all three cases. (B) Confocal sections of representative mosquito midgut epithelial sheets infected with <i>GFPp</i>, <i>NcPIS_3</i>, ANKA or <i>NcPIS_1</i> parasites, and stained with antibodies against the mosquito serpin6 (SRPN6) (red) and the ookinete surface protein P28 (green). Nuclei are stained with TO-PRO 3 (blue). Arrows indicate the position of ookinetes. <i>NcPIS_1</i> and <i>3</i> ookinetes fail to induce an epithelial response, which in contrast is revealed in the case of ANKA and <i>GFPp</i> infected midguts by SRPN6 specific antibody. Scalebar 10μM. (C) Confocal microscopy of mature oocysts. The two types of <i>NcPIS_3</i> oocysts (upper panel) are shown alongside the single oocyst that showed a normal morphology 14 days post infection. Main oocyst morphology includes: Normal sized oocysts with diffused nuclear material and a collapsed capsule (CO) and oocysts arrested in an early developmental stage (minute oocysts: MO). Green corresponds to GFP expression; nuclei are stained with TO-PRO 3. (Lower panel) <i>NcPIS_1</i> abnormal oocysts labeled with the antibody recognizing the oocyst capsule protein PbCap380. Scale bar 20μM.