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Testing Possible Causes of Gametocyte Reduction in Temporally Out-Of-Synch Malaria Infections

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Testing Possible Causes of Gametocyte Reduction in Temporally Out-Of-Synch Malaria Infections

Mary L Westwood et al. Malar J.

Abstract

Background: The intraerythrocytic development cycle (IDC) of the rodent malaria Plasmodium chabaudi is coordinated with host circadian rhythms. When this coordination is disrupted, parasites suffer a 50% reduction in both asexual stages and sexual stage gametocytes over the acute phase of infection. Reduced gametocyte density may not simply follow from a loss of asexuals because investment into gametocytes ("conversion rate") is a plastic trait; furthermore, the densities of both asexuals and gametocytes are highly dynamic during infection. Hence, the reasons for the reduction of gametocytes in infections that are out-of-synch with host circadian rhythms remain unclear. Here, two explanations are tested: first, whether out-of-synch parasites reduce their conversion rate to prioritize asexual replication via reproductive restraint; second, whether out-of-synch gametocytes experience elevated clearance by the host's circadian immune responses.

Methods: First, conversion rate data were analysed from a previous experiment comparing infections of P. chabaudi that were in-synch or 12 h out-of-synch with host circadian rhythms. Second, three new experiments examined whether the inflammatory cytokine TNF varies in its gametocytocidal efficacy according to host time-of-day and gametocyte age.

Results: There was no evidence that parasites reduce conversion or that their gametocytes become more vulnerable to TNF when out-of-synch with host circadian rhythms.

Conclusions: The factors causing the reduction of gametocytes in out-of-synch infections remain mysterious. Candidates for future investigation include alternative rhythmic factors involved in innate immune responses and the rhythmicity in essential resources required for gametocyte development. Explaining why it matters for gametocytes to be synchronized to host circadian rhythms might suggest novel approaches to blocking transmission.

Keywords: Chronoimmunology; Conversion rate; Inflammatory cytokine; Innate immunity; Intraerythrocytic development cycle; Malaria; Phenotypic plasticity; Plasmodium; Reproductive effort; TNF-α.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
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 Per1/2(−/−) 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”
Fig. 2
Fig. 2
Conversion estimates, alongside mean ± SE (calculated post-transformation), for parasites in- and out-of-synch with host circadian rhythms. Points represent raw data, log10-transformed to approximate normality. Data from experiment in O’Donnell et al. [6]
Fig. 3
Fig. 3
TNF concentrations (pg/ML) alongside mean ± SE at 2 HPI, after treatment at either ZT0 (lights-on) or ZT12 (lights-off) for two doses of TNF (30 μg/kg or 60 μg/kg). Points represent raw data
Fig. 4
Fig. 4
a Cumulative gametocyte density (gametocytes/mL blood) for all sampling timepoints, alongside mean ± SE (calculated post-transformation), in mice injected with either TNF or carrier at either ZT0 or ZT12. b Gametocyte density (gametocytes/mL blood), alongside mean ± SE, for combined treatment groups (TNF and control) in mice injected at either ZT0 or ZT12 for each sampling time (− 1 (baseline), 2-, and 12 HPI). In both plots, points represent raw data, square-root-transformed to approximate normality, and scaled to have a mean of 0 and a standard deviation of 1
Fig. 5
Fig. 5
Gametocyte density (gametocytes/mL blood), alongside mean ± SE (calculated post-transformation), averaged across all sampling timepoints (4-, 8-, and 12-HPI) for gametocytes treated at ages 19-, 25-, and 31-h old and according to whether they were treated with TNF or PBS carrier only. Points represent raw data, square root-transformed to approximate normality, and scaled to have a mean of 0 and a standard deviation of 1

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