Fig. 5. Triple immunofluorescence of microtubules (green), IMC-1 (red) and DNA (blue) in samples treated for 48 hours with 0.5 µM oryzalin (A,B) or 2.5 µM oryzalin (C-E). Tachyzoites treated with 0.5 µM oryzalin undergo nuclear division and bud as round parasites (A). 48 hours after drug removal, these parasites continue replicating as crescent-shaped parasites with correctly sized and segregating nuclei and fully restored subpellicular microtubules (B). Tachyzoites treated with 2.5 µM oryzalin continue to grow (this is a single parasite) but fail to undergo nuclear division or bud off daughter parasites (C). When these samples are removed from the drug for 48 hours, they attempt to bud as multiple daughter parasites in a process reminiscent of schizogeny (D). The large, polyploid nuclear mass (large arrow) cannot be segregated correctly, so daughter buds (small arrows) are completely devoid of nuclear material or contain the apicoplast. Anucleate zoids (E1-4) can complete budding, escape from the vacuole and in some cases reinvade new cells but are incapable of growth or replication. E1 and 2 show triple-labeled zoids and E3 and 4 show the same cells with the tubulin staining omitted for clarity. The zoid in E1/3 has an apicoplast but lacks a nucleus, as does the left-hand zoid in E2/4. The right-hand zoid in E2/4 lacks both nucleus and apicoplast.