Plasmodium Ca2+ Oscillation in the Blood Stage Figure 1. Life cycle of Plasmodium. falciparum. Schematic illustration of the life cycle of P. falciparum. The blood stages on which this study is focused are shown in detail. doi:10.1371/journal.pone.0039499.g001 signalling controls various vital functions such as protein secretion, motility, cell invasion and differentiation [7–15]. With regard to intracellular Ca2+ signalling in Plasmodium species, numerous studies have focused on calcium-dependent protein kinases, which are activated downstream of Ca2+ release from intracellular Ca2+ stores, as an important therapeutic target for antimalarial drug development [8,9,12,15,16]. Particularly in the blood stage, Ca2+ has been considered to be a key regulator of the parasite egress and invasion of erythrocytes [16–18]; however, little is known about the role of Ca2+ signalling in intraerythrocytic development of Plasmodium species. In this study, we observed the intracellular dynamics of Ca2+ throughout the intraerythrocytic stages of the FCR-3 strain of P. falciparum and found that stage-specific spontaneous Ca2+ oscillations which can be blocked by the inositol 1,4,5-trispho- sphate (IP3) receptor inhibitor 2-aminoethyl diphenylborinate (2- APB) occur in the ring form and trophozoite. Examination of the effects of 2-APB on the in vitro intraerythrocytic parasite development and electron microscopic observations revealed that blockage of Ca2+ oscillations caused severe degeneration and breakdown of successive asexual reproduction in the intraeryth- rocytic parasites, resulting in death of them. Furthermore, 2-APB showed a similar effect against the chloroquine-resistant K1 strain of P. falciparum. Results and Discussion IP3-induced Ca2+ Oscillation in P. falciparum Figure 2 and S1 shows fluorescence Ca2+ images of each intraerythrocytic developmental stage of the FCR-3 strain: early ring forms (ERf, parasites with smaller cell size than trophozoite without malaria pigment), late ring forms (LRf, parasites with cell size between early ring form and trophozoite without malaria pigment), early trophozoites (ET, parasites with a single nucleolus, malaria pigment and immature food vacuole), late trophozoites (LT, parasites with a single nucleolus and mature food vacuole), schizonts (S, parasites with multiple nuclei) and merozoites (M). Ca2+ imaging of parasites was performed in culture chambers at 37uC in an atmosphere of 5% O2 and 5% CO2, conditions identical to those in conventional in vitro parasite culture. The Fluo-4 fluorescence in a parasite cytoplasm (F) was calculated by subtraction of the background fluorescence and normalized to the minimum fluorescence during the imaging period (Fmin). In early ring forms (ERf) and early trophozoites (ET), spontaneous Ca2+ oscillations were observed (Fig. 2A and B, left). Dimethyl sulfoxide (DMSO) was used as a solvent control. The frequency of Ca2+ oscillations was higher in early ring forms than that in early trophozoites. The subcellular distribution of Fluo-4 in the early trophozoites indicates that free Ca2+ were evenly distributed in the cytoplasm (Fig. 2B), whereas in the late trophozoites with mature food vacuole, Ca2+ gradient between the digestive food vacuole and cytoplasm, similar to that previously reported [19,20] was observed being independent of the addition of 2-APB (Fig. S2). 2- APB was a well-established inhibitor of IP3 receptor/Ca2+ channels developed in our previous study [21,22] and the blockage of melatonin-induced Ca2+ release by 2-APB in P. falciparum has been demonstrated [23]. Treatment with 100 mM 2-APB almost completely blocked Ca2+ oscillations (Fig. 2A and B, right). On the other hand, in the late ring forms (LRf), late trophozoites (LT), schizonts (S) and merozoites (M), small periodic Ca2+ fluctuations were observed, and notable effects of 2-APB were not detected (Fig. S1). To investigate the effects of 2-APB in detail, we performed quantitative analysis of the effect of 2-APB on the amplitude of periodic Ca2+ fluctuations in late ring form, late trophozoite, schizont and merozoite stages. The mean amplitude was calculated by subtracting the mean minimal value of F/Fmin from its mean maximal value. A statistically significant effect of 2- APB was observed in merozoites (*, P = 0.0116, two-tailed unpaired t test) (Fig. S3). This result supports those of previous studies demonstrating that an increase in cytosolic Ca2+ concen- tration is involved in the regulation of merozoite invasion of the erythrocytes [16–18]. In apicomplexan parasites, Ca2+ transients and oscillations have been reported to be evoked by several inducers [10,13,24,25]; however, our study is the first to demonstrate spontaneous Ca2+ oscillations, i.e. without the addition of exogenous inducers. Blockage of spontaneous Ca2+ oscillations by 2-APB in early ring forms and early trophozoites strongly suggests that the observed Ca2+ oscillations are regulated by a putative IP3 receptor/Ca2+ channel that is activated by IP3 binding. In Plasmodium the phospholipase C (PLC) pathway is known to be involved in the release of Ca2+ from the intracellular Ca2+ store, PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e39499