Plasmodium Ca2+ Oscillation in the Blood Stage Figure 5. Electron micrographs of parasites treated with 2-APB. (A) After 30 h of DMSO administration (control group; original magnification, 630,000). (B) After 30 h of 100 mM 2-APB administration (original magnification, 630,000). (C) After 30 h of 100 mM 2-APB administration (original magnification, 630,000). (D) After 30 h of 100 mM 2-APB administration. Reticular ER structure is observed (arrow) (original magnification, 650,000). (E) After 40 h of 100 mM 2-APB administration. The nuclear envelope (NE) surrounded by ribosomal granules (Ri) is observed (arrow) (original magnification, 650,000). (F) After 40 h of 100 mM 2-APB administration. Formation of rhoptries (Rh) and other micro-organelles is observed (original magnification, 650,000). N, nucleus; MP, malaria pigment; MC, Maurer’s cleft. doi:10.1371/journal.pone.0039499.g005 togenesis of P. falciparum [45–49] and interplay between cAMP and Ca2+ as second messengers was also reported [24]. Taken together, our results suggest the possibility that Ca2+ oscillations during ring form stage might be involved in triggering gameto- cytogenesis, rather than in maintaining asexual erythrocytic cycle. During the erythrocytic cycle, Plasmodium species repeat the drastic morphological and functional changes; invasion, feeding, multiplication, secretion and structural modification of the parasite-infected erythrocytes. Our study clearly indicated that Ca2+ signaling plays a pivotal role for cell growth and differen- tiation of P. falciparum, suggesting that it could be a useful experimental model organism for understanding fundamental roles and mechanisms of Ca2+ signaling conserved from unicellular organisms to humans. In clinical aspects, the mature-stage of P. falciparum modifies the surface structure of the host erythrocytes. The parasite-infected erythrocytes adhere to endothelium and sequester the microvasculature of several organs and block the blood circulation. This pathology called ‘‘sequestration’’ causes severe symptoms including coma, acute respiratory distress, kidney failure and death in humans. Thus, consequences of malaria are closely associated with intraerythrocytic P. falciparum development. Although functional evidence for IP3-induced Ca2+ release has been reported in Plasmodium species [50,51], little attention has been paid to the molecules upstream of Ca2+ release from intracellular Ca2+ stores due to the lack of molecular identities for them. An IP3R gene, as defined in metazoans, has not been identified in the Plasmodium genome. This apparent absence could be due to the lack of homology with IP3Rs in metazoans as in plants [26,52]. Our results increase the possibility that the identification of molecules responsible for generating Ca2+ oscillations including IP3R will provide promising targets for the development of novel antimalarial drugs. Materials and Methods P. falciparum Culture The FCR-3 and K1 strains of P. falciparum were cultured using the modified method of Trager and Jensen [53] in RPMI medium (Invitrogen/Gibco) supplemented with 0.5% Alubumax I (Invitro- gen), 25 mM HEPES, 24 mM sodium bicarbonate, 0.5 g/L L- glutamine, 50 mg/L hypoxanthine, 25 mg/mL gentamicin (Sigma) and human erythrocytes (from a healthy Japanese volunteer) at a Figure 6. Effect of 2-APB on ER structure. The parasite nucleus and ER were stained simultaneously with Hoechst 33342 (blue) and ER-Tracker (red) after 30 h of DMSO (upper panels) or 2-APB (lower panels) administration. Merged images are shown in the right columns (Merge). doi:10.1371/journal.pone.0039499.g006 PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e39499