Plasmodium Ca2+ Oscillation in the Blood Stage Figure 4. Time window for inhibition of intraerythrocytic P. falciparum development by 2-APB. (A) 2-APB was removed at 10 h of the assay. (B) 2-APB was removed at 21 h of the assay. (C) 2-APB was added at 21 h of the assay. (D) Percentages of T and S in 2 independent experiments shown in (C) just before 2-APB was added at 21 h of the assay. (E) 2-APB was added at 28 h of the assay. Parasitaemia at 40 h of the assay is shown as mean + S.D. of 3 independent counts of 3 wells. Stages with parasitaemia of less than 0.1% are not shown. The difference in Rf parasitaemia between the DMSO and 2-APB groups was analysed statistically (two-tailed unpaired t test ) and P values are given in each panel (A, B, C and E). (F) Effects of 100 mM 2-APB on the area, perimeter and maximum diameter of parasites. Three experimental groups were tested as follows. 2-APB was added at the beginning of the assay during synchronization, and cell size was analysed at 10 or 20 h of the assay. 2-APB was added at the beginning of the assay, removed at 10 h of the assay and cell size was measured at 20 h of the assay. Fifty parasites were measured in each experimental group. P values compared with DMSO controls are given in each panel (two-tailed unpaired t test with Welch’s correction). doi:10.1371/journal.pone.0039499.g004 that between trophozoite to schizont stages. Considering the reversible effect of 2-APB on the early ring form stage (Fig. 4F), we conclude that the lethal effect of 2-APB on intraerythrocytic parasite development was caused mainly by the blockage of Ca2+ oscillations in the early trophozoite stage. The importance of Ca2+- mediated signals for merozoites invasion of erythrocytes has been reported previously [17,18], and hence, the results in Fig. 4E suggest that IP3-induced periodic Ca2+ fluctuations in the merozoites stage has an important role in parasite invasion. Moreover, the weak effect of 2-APB on strain K1 at 24 h of the assay (Fig. 3E) may be attributable to the fact that most of the K1 parasites did not reach the stage (trophozoite to early schizont stage) at which 2-APB had a lethal effect in the FCR-3 strain (Fig. 4). However, it remains possible that the effectiveness of 2- APB in the ring form stage of strain K1 differs from its effectiveness in the FCR-3 strain. Severe Parasite Degeneration Caused by 2-APB Ultrastructural changes induced by 2-APB were observed by transmission electron microscopy. As shown in Fig. 5A and Fig. S8, parasites cultured with DMSO at 30 h of the assay maintained a normal structure. In contrast, highly dense chromatin masses in the nucleus and highly dense degeneration were consistently observed 30 h after the assay (Fig. 5B and C). The formation of Maurer’s cleft and malaria pigment in the food vacuoles suggests that degeneration induced by 2-APB occurred after intraerythro- cytic development to some extent (Fig. 5B). In Plasmodium species, the nuclear envelope is considered the main ER compartment [31–34]. We confirmed that the ER-Tracker signals surrounded the nuclei of parasites stained blue with Hoechst 33342 and cultured with DMSO, whereas in parasites cultured with 2-APB the ER-Tracker signals became broad and extended to the cytosol (Fig. 6). Similarly, in electron micrographs parasites cultured with 2-APB characteristically showed a dilated nuclear envelope and ER (Fig. 5B and C) and the dilated nuclear envelopes connected with the dilated ER, forming a reticular ER structure (Figs 5D and 6). In a previous report, a nucleus surrounded by rough ER in a late schizont with the nuclear envelope bearing numerous ribosomal granules was observed by electron microscopy [35] and similar electron micrographs were obtained with parasites cultured with DMSO in this study (Fig. S8B). Interestingly, parasites cultured with 2-APB frequently exhibited an increased number of ribosomal granules, distributed at even intervals in a line along the dilated nuclear envelope (Fig. 5E). Similar morphological alterations in the nuclear envelope and rough ER were also observed in some parasites exposed to artemisinin [36], which is known to inhibit the sarco/endoplasmic reticulum Ca2+- ATPase orthologue (PfATP6) of P. falciparum [37], suggesting that these ultrastructural changes are commonly induced by interfer- ence with Ca2+ signalling. Dilatation of the nuclear envelope and ER is possibly among the features of cell degeneration, but it may also compensate for disrupted Ca2+ signalling, since ER is a multifunctional and highly dynamic organelle, undergoing con- stant movement and reorganization depending on cellular conditions [38,39]. Although most of the parasites cultured with 2-APB showed severe degeneration (Fig. 5B and C), schizonts in which merozoites with normal micro-organelles were formed were also present (Fig. 5F). At 40 h of the assay, the number of merozoites formed in each schizont of parasites cultured with 2- APB was significantly smaller than that formed in parasites cultured with DMSO (Fig. S9A and B), suggesting that parasite development was inhibited by 2-APB even in parasites in which merozoites were formed normally. Conclusive Remarks In higher eukaryotes, disrupted intracellular Ca2+ signalling and accumulation of aberrant proteins is known to cause ER stress, a hallmark of cell death that is associated with many neurodegen- erative diseases [40–43]. However, very little is known about the biological significance and molecular composition of Ca2+ signaling systems in unicellular organisms. In this study, we clearly showed the stage-specific spontaneous Ca2+ oscillations in the intraerythrocytic stages of P. falciparum, a unicellular eukaryote, without any exogenous extracellular stimulation. We further demonstrated that the blockage of these Ca2+ oscillations by 2- APB caused severe cellular degeneration resulting to the death of the parasites. The half maximal inhibitory concentration (IC50) value of 2-APB for inhibition of IP3-induced Ca2+ release in mammalian cells was ,40 mM [21]. Thus, a high concentration (100 mM) 2-APB was used in this study. Such a high dose 2-APB possibly exerts pleiotropic effects on both host cells and parasites. However, our findings in this study strongly support the idea that the severe inhibitory effect of 2-APB on the intraerythrocytic development of the parasites is primarily due to the specific blockage of Ca2+ oscillations of the early trophozoites. First, the normal development of the parasites was observed in 2-APB- pretreated erythrocytes (Fig. S5). Secondly, the critically effective time window of 2-APB on the intraerythrocytic development of the parasites was coincident with the early trophozoite stage in which spontaneous Ca2+ oscillation was observed (Fig. 2, 4 and S7). Thirdly, the delayed development of the parasites was recovered when 2-APB was removed at 10 h of the assay (Fig. 4F). Lastly, 100 mM 2-APB did not disrupt Ca2+ gradient between the food vacuole and cytoplasm unlike a mM order of chloroquine treatment permeabilized the food vacuole membrane, resulting in the cell death [20]. Interestingly, a much higher contribution of the Ca2+ oscillations in trophozoite stage to the intraerythrocytic development of the parasites was observed than that in the ring form stage. This result indicates that the Ca2+ oscillation observed in ring form stage has a different physiological role from that in the trophozoite stage. In Plasmodium species, gametocytogenesis delivers the sexual-stage of the parasite known as gametocyte involved in the transmission from the mammalian host to the mosquito. Gametocyte development can be induced by host factors or drug treatment, and of which signal transduction pathways are involved in this process [44]. There is consistent evidence that phorbol ester inducing pathways and cyclic AMP (cAMP) signalling pathway are involved in triggering gametocy- PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e39499