Plasmodium Ca2+ Oscillation in the Blood Stage Figure 2. Cytosolic calcium (Ca2+) dynamics in the early ring forms (ERf) (A) and early trophozoites (ET) (B) and effects of 2- aminoethyl diphenylborinate (2-APB). Each colour represents cytosolic Ca2+ dynamics acquired from individual parasites in the presence (right columns) or absence (left columns) of 100 mM 2-APB. Embedded images in left panels are representative images of Fluo-4-loaded P. falciparum during each intraerythrocytic stage (indicated by arrowheads). Scale bars, 5 mm. doi:10.1371/journal.pone.0039499.g002 and the effectiveness of U73122, a commonly used PLC inhibitor, has been demonstrated [25]. PLC catalyses the hydrolysis of 1- phosphatidyl-D-myo-inositol-3,4,5-trisphosphate to the second messenger molecules diacylglycerol and IP3. As shown in Fig. S4A, after 5 min of pre-treatment with 10mM U73122, Ca2+ oscillations in early ring forms (ERf, left) and early trophozoites (ET, right) were almost completely inhibited. In apicomplexan parasites, two intracellular Ca2+ stores are known to be involved in Ca2+ release: the endoplasmic reticulum (ER) and acidocalcisomes including the food vacuole [26,27]. To investigate the source of the observed Ca2+ oscillations, we depleted Ca2+ stores before imaging by using thapsigargin (Tg), a specific inhibitor of sarco/endoplas- mic reticulum Ca2+–ATPase, and concanamycin A (CMA), a specific inhibitor of vacuolar-type H+-ATPase. The effects of these inhibitors have been demonstrated in Plasmodium species [28–30]. We also confirmed the effectiveness of these compounds in depleting Ca2+ by Ca2+ imaging in early ring forms and early trophozoites using perfusion experiments (Fig. S4B and C). As shown in Fig. S4D, Ca2+ depletion by pre-treatment with 2 mM Tg for 30 min significantly reduced Ca2+ oscillations in early ring forms (ERf, left) and early trophozoites (ET, right). In contrast, Ca2+ depletion by pre-treatment with 100 nM CMA had no effect on Ca2+ oscillations in early ring forms (ERf, left) and early trophozoites (ET, right) (Fig. S4E). In these two stages the digestive food vacuole, which is known to be a Ca2+ pool sensitive to both thapsigargin and a vacuolar-type H+-ATPase inhibitor, bafilomy- cin A1 [19], is not formed. Taken together, these results indicate that spontaneous IP3-induced Ca2+ release from a thapsigargin- sensitive Ca2+ store, ER occurred in early ring forms and early trophozoites during intraerythrocytic P. falciparum development. Next, we investigated the effects of 2-APB on the intraerythrocytic P. falciparum development to understand the physiological roles of these spontaneous IP3-induced Ca2+ oscillations. 2-APB Blocks P. falciparum Development The effects of 2-APB on intraerythrocytic development were investigated using synchronized parasite cultures in the ring form stage, with initial parasitaemia of approximately 1%. Figure 3A shows parasitaemia of each developmental stage at 20, 30 and 40 h of the assay. In the presence of 100 mM 2-APB intraeryth- rocytic development of the parasites was delayed compared to that in the presence of dimethyl sulphoxide (DMSO). Figure 3B shows the morphology of intraerythrocytic parasites. Parasites cultured with DMSO developed into early schizonts (parasites with fewer than 8 nuclei) at 20 h of the assay (Fig. 3B, panel 1). These schizonts developed into healthy late schizonts (parasites with at least 8 nuclei) and produced ring forms in the next developmental cycle at 30 and 40 h of the assay (Fig. 3B, panels 2 and 3). In contrast, parasites cultured with 2-APB remained at the tropho- zoite stage (parasites with a single nucleus) with abnormal morphology at 20 h of the assay (Fig. 3B, panel 4). These abnormal trophozoites could develop into early schizonts and late schizonts but exhibited abnormal morphology at 30 and 40 h of the assay (Fig. 3B, panels 5 and 6). To quantitatively examine the effect of 2-APB on intraerythrocytic parasite development, the area, perimeter and maximum diameter of the parasites were analysed at 15, 30 and 40 h of the assay. At 40 h of the assay only schizonts were analysed. At 15, 30 and 40 h of the assay, parasites PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e39499