(A) Time course of parasite growth of untreated (blue), clindamycin (green), pyrimethamine (black), or actinonin (red) treated parasites over the course of two lytic cycles. Unlike P. falciparum, T. gondii undergoes multiple replication cycles in the host cell before lysis, thus each T. gondii lytic cycle represents multiple parasite replication cycles. Pyrimethamine inhibits parasite dihydrofolate reductase and was used as a control for a non-apicoplast targeting drug that inhibits growth in a single lytic cycle. As previously reported (Camps et al., 2002), clindamycin, an apicoplast translation inhibitor, gave a ‘delayed death’ phenotype in T. gondii characterized by completed cell divisions and host re-invasion during drug treatment in the first lytic cycle, followed by halted cell division in the second lytic cycle. Actinonin also led to growth inhibition in the second lytic cycle, suggesting that it also targets the apicoplast. Error bars represent the SEM of two biological replicates. It is important to distinguish between apicoplast-associated ‘delayed death’ in P. falciparum and that in T. gondii. In T. gondii, apicoplast loss occurs in the first lytic cycle and is temporally separate from defects in parasite cell division and growth inhibition observed in the second lytic cycle; whereas in P. falciparum defects in apicoplast biogenesis, parasite cell division, and growth inhibition all occur in the second replication cycle. ‘Delayed death’ in T. gondii therefore appears more broadly associated with disruption of apicoplast biogenesis, where ‘delayed death”in P. falciparum appears more specific to disruption of apicoplast gene expression that leads to delayed biogenesis defects. (B) To determine dose-dependent drug target specificity, we used a previously described fluorescent growth assay to quantify the kinetics of death in T. gondii (Gubbels et al., 2003). Briefly, T. gondii expressing tandem-tomato are treated with drug and fluorescence is quantified daily as a proxy of parasite replication. While 40 uM actinonin leads to delayed-death kinetics similar to clindamycin, 80 uM actinonin results in immediate death kinetics similar to pyrimethamine, indicating that at this concentration, actinonin hits a second, non-apicoplast target.