Plasmodium Ca2+ Oscillation in the Blood Stage Figure 3. Inhibition of intraerythrocytic P. falciparum development by 2-APB. (A) The FCR-3 strain was cultured for 40 h of the intraerythrocytic development cycle. Cultures were terminated at 20, 30 and 40 h of the assay after synchronization, and thin smears of erythrocytes were prepared for parasite counting. Representative results of 3 independent experiments are shown. (B) Morphology of intraerythrocytic parasites cultured with DMSO or 100 mM 2-APB at 20, 30 and 40 h of the assay after synchronization. (C) 100 mM 2-APB significantly decreased the area, perimeter and maximum diameter of intraerythrocytic parasites at 15, 30 and 40 h of the assay after synchronization. Columns and error bars represent the mean + S.D. Fifty parasites were measured at each time point. The P values compared with DMSO controls are given below each figure (two-tailed unpaired t test with Welch’s correction). (D) Cultures were terminated at 40 and 70 h of the assay after synchronization for parasite counting. Culture medium with DMSO or 2-APB was replaced at 40 h. Representative results of 3 independent experiments are shown. (E) The chloroquine-resistant strain K1 was cultured for 72 h of the intraerythrocytic development cycle. Cultures were terminated at 24, 48 and 72 h of the assay after synchronization, and thin smears of erythrocytes were prepared for parasite counting. Culture medium with DMSO or 2-APB was replaced at 24 and 48 h. Representative results of 3 independent experiments are shown. Parasiaemia of ring form (Rf), trophozoites (T), early schizonts (ES) and late schizonts (LS) is shown as mean + S.D. of 3 independent counts of a single well (A) or 3 wells (D, E). Stages with parasitaemia of less than 0.1% are not shown (A, D and E). doi:10.1371/journal.pone.0039499.g003 cultured with 2-APB showed significantly decreased size compared to those cultured with DMSO, indicating that 2-APB delayed intraerythrocytic parasite development (Fig. 3C). Furthermore, analysis of the parasite size revealed that the increases in the 3 parameters were terminated at 30 h after 2-APB treatment, suggesting that the critical time of the effect of 2-APB is approximately 30 h. To investigate the fate of abnormal schizonts at 40 h of the assay in which parasites were cultured with 2-APB (Fig. 3B, panel 6), the assay was prolonged for another 30 h (Fig. 3D). Results at 40 h of the assay confirmed those shown in Fig. 3A. At 70 h parasites cultured with 2-APB developed into a few late schizonts or ring forms with abnormal morphology (1 in 5000–8000 erythrocytes), although those cultured with DMSO developed normally into late schizonts or ring forms in the next developmental stage. On the other hand, parasites could develop normally in 2-APB-pretreated erythrocytes similar to that in cultures with DMSO-pre-treated erythrocytes (Fig. S5). This indicates that the effect of 2-APB is not due to the disruption of erythrocyte physiology, which is significant to intraerythrocytic parasite development and invasion. From these results we conclude that 2-APB directly inhibits intraerythrocytic parasite development by blocking its normal cell cycle, resulting in a failure to maintain the successive developmental stages of asexual blood forms. 2-APB Acts on a Chloroquine-resistant Strain Next, we examined the effect of 2-APB on the intraerythrocytic development of the chloroquine-resistant strain K1 of P. falciparum using synchronized parasite cultures in the ring form stage with initial parasitaemia of approximately 2%. At 24 h of the assay a tendency towards decreasing trophozoite parasitaemia was repro- ducibly observed in parasites cultured with 100 mM 2-APB (Fig. 3E, 24 h). The slight inhibitory effect of 2-APB at 24 h of the assay was confirmed by measuring the area, perimeter and maximum diameter of the parasites (Fig. S6). At 48 h of the assay, intraerythrocytic parasite development in the presence 2-APB was delayed compared to that in the presence of DMSO, similar to that observed in the FCR-3 strain (Fig. 3E, 48 h). A further 24 h of the assay revealed that the number of infected erythrocytes in the presence of 2-APB was much lesser than that in the presence of DMSO, in which there was a high level of parasitaemia (Fig. 3E, 72 h). Trophozoites are the Main Target of 2-APB To investigate the stages of intraerythrocytic development that 2-APB may block, 2-APB was removed from or added to cultures at different time intervals and parasitaemia was determined after 40 h of the assay for each developmental stage. Two independent results are shown in Fig. 4A-E. When 2-APB was removed from culture at the ring form stage (10 h of the assay) (Fig. 4A), parasites present at 40 h of the assay developed into schizonts with normal morphology, but ring form parasitaemia in the next developmen- tal cycle was significantly decreased. On the other hand, when 2- APB was removed between trophozoite to early schizont stages (21 h of the assay) (Fig. 4B), parasites present at 40 h of the assay developed into schizonts with either normal or abnormal morphology, and the effect of 2-APB in decreasing ring form parasitaemia in the next developmental cycle was greater than that shown in Fig. 4A. When 2-APB was added at the same time (21 h of the assay) (Fig. 4C), parasites present at 40 h of the assay developed into schizonts with abnormal morphology and ring form parasitaemia in the next developmental cycle was signifi- cantly decreased. These results indicate that the time at which 2- APB is most effective in blocking intraerythrocytic parasite development is between the trophozoite to early schizont stages. This is supported by the observation that a higher percentage of trophozoites at the time of addition of 2-APB resulted in greater effects on ring form parasitaemia (Fig. 4D). Furthermore, when 2- APB was added at a later stage (late schizont stage, 28 h of the assay), the parasites were able to develop into fully mature schizonts with segmented merozoites and produce ring forms like those observed in the control culture (Fig. 3B); however, ring form parasitaemia at 40 h of the assay was significantly decreased (Fig. 4E). This result suggests that 2-APB affects late schizont maturation and erythrocyte egress and/or invasion of merozoites. Finally, to further investigate the effect of 2-APB on the ring form stage, parasite size was measured at 10 and 20 h of the assay (Fig. 4F). In parasites cultured with DMSO, increases in area, perimeter and maximum diameter at 20 h of the assay were higher than those observed at 10 h of the assay as expected from the results in Fig. 3C. A similar result was obtained when DMSO was removed at 10 h of the assay. In contrast, in parasites cultured with 2-APB at 10 and 20 h of the assay, all 3 parameters were significantly smaller than those in parasites cultured with DMSO; however, when 2-APB was removed at 10 h of the assay, all 3 parameters were slightly smaller than those in parasites cultured with DMSO but recovered to a level at which no statistically significant difference was detected. From these results we propose 2 possibilities to explain the finding that removal of 2-APB at 10 h of the assay caused a significant decrease in ring form parasitaemia at 40 h of the assay (Fig. 4A). (1) The growth of trophozoites that were exposed to 2-APB before 10 h of the assay was inhibited. and (2) a proportion of ring forms did not recover and resume normal development. Together with results of Ca2+ imaging experiments (Fig. 2), the inhibitory effects of 2-APB on Ca2+ oscillations and intraerythrocytic parasite development in the blood stage are summarized in Fig. S7. Ca2+ oscillations observed in early ring forms and early trophozoites reduced on treatment with 2-APB (Fig. 2A and B), but treatment at the ring form stage showed a significantly weaker effect on intraerythrocytic development than PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e39499