Three photoschedules were used to generate temporally-staggered cohorts of gametocytes simultaneously perturbed at different ages. Parasites were collected from donor mice at their ZT0, allowing infections in experimental mice to be staggered by 6-hours so that at the same times GMT, all infections could be sampled and treated with TNF or PBS yet, different ages of gametocytes (19-, 25-, and 31-hours-old) could be targeted. By using (−/−) mice housed in constant darkness as the experimental hosts, the relevance of gametocyte age was decoupled from the canonical host circadian-clock-controlled rhythms. The ages of focal gametocyte cohorts (labelled “gametocyte age (h)”, defined as hours post RBC invasion) at each sampling event and treatment (in GMT) are highlighted in bold and the ages of the previous (“younger”) and subsequent (“older”) cohorts are illustrated with faint text. Immature gametocytes not yet detectable via microscopy are denoted by “ND”, and gametocytes not yet produced are denoted by “NA”
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