Figure 4

Phenotypic analysis of pycpw-wpc-1 gene disruption in parasites. (A) Schematic representation of pycpw-wpc-1 gene disruption. To investigate the function of PyCPW-WPC-1 during sexual-stage development, the pycpw-wpc-1 coding region was disrupted by insertion of the hdhfr cassette using double-crossover homologous recombination. (B) Depletion of PyCPW-WPC-1 protein in transgenic parasites. Ookinete antigens were prepared from pycpw-wpc-1-disrupted parasites and wild-type parasites. Western blotting using specific antibodies demonstrated the obvious reduction in PyCPW-WPC-1 protein expression as compared to the reference band from wild-type ookinete extracts (arrow). (C) Disappearance of the PyCPW-WPC-1 surface-localizing pattern in transgenic ookinetes. Pycpw-wpc-1 gene-disrupted ookinetes appeared normal in morphology, as shown by DIC images (left panels) and surface localization of Pys25 (right panels). Compared to the wild-type parasite, pycpw-wpc-1 gene-disrupted ookinetes exhibited little or no surface expression of PyCPW-WPC-1. The bar indicates 5 μm. (D) pycpw-wpc-1-disrupted parasites showed no defects in oocyst formation. The oocyst numbers were counted from individual midguts dissected from mosquitoes, which were fed on mice infected with wild-type or pycpw-wpc-1-disrupted parasites. Horizontal lines indicate mean oocyst numbers.