Malarial fever arises from the synchronous bursting of human red blood cells by the Plasmodium parasite. The released parasites re-infect neighboring red blood cells and undergo another asexual cycle of differentiation and proliferation for 48 hours, before again bursting synchronously. The synchrony of bursting is lost during in vitro culturing of the parasite outside the human body, presumably because the asexual cycle is no longer entrained by host-specific circadian cues. Therefore, most in vitro malaria studies have relied on the artificial synchronization of the parasite population. However, much remains unknown about the degree of timing heterogeneity of asexual cycles and how artificial synchronization may affect this timing. Here, we combined time-lapse fluorescence microscopy and long-term culturing to follow single cells and directly measure the heterogeneous timing of in vitro asexual cycles. We first demonstrate that unsynchronized laboratory cultures are not fully asynchronous and the parasites exhibit a bimodal distribution in their first burst times. We then show that synchronized and unsynchronized cultures had similar asexual cycle periods, which indicates that artificial synchronization does not fundamentally perturb asexual cycle dynamics. Last, we demonstrate that sibling parasites descended from the same schizont exhibited significant variation in asexual cycle period, although smaller than the variation between non-siblings. The additional variance between non-siblings likely arises from the variable environments and/or developmental programs experienced in different host cells.

We used timelapse fluorescence microscopy of GFP-expressing P. falciparum to quantify the timing and variability of the asexual cycle in in vitro culturing conditions.  We measured the timing of GFP-parasite bursting across two asexual cycles for unsynchronized and sorbitol-shock synchronized cultures (two biological replicates).  Dataset consists of one example timelapse fluorescence movie of GFP-labelled parasites (Movie S1), data of first-burst time, second-burst time, and asexual cycle time of synchronized and unsynchronized parasites for both biological replicates (Dataset S1), and timecourse data of ring, trophozoite, schizont counts for a synchronized GFP culture released into control and light condition (Dataset S2).