Despite evidence that γδ T cells play an important role during malaria, their precise role remains unclear. During murine malaria induced by Plasmodium chabaudi infection and in human P. falciparum infection, we found that γδ T cells expanded rapidly after resolution of acute parasitemia, in contrast to αβ T cells that expanded at the acute stage and then declined. Single-cell sequencing showed that TRAV15N-1 (Vδ6.3) γδ T cells were clonally expanded in mice and had convergent complementarity-determining region 3 sequences. These γδ T cells expressed specific cytokines, M-CSF, CCL5, CCL3, which are known to act on myeloid cells, indicating that this γδ T cell subset might have distinct functions. Both γδ T cells and M-CSF were necessary for preventing parasitemic recurrence. These findings point to an M-CSF-producing γδ T cell subset that fulfills a specialized protective role in the later stage of malaria infection when αβ T cells have declined.

Infected hosts differ in their responses to pathogens; some hosts are resilient and recover their original health, whereas others follow a divergent path and die. To quantitate these differences, we propose mapping the routes infected individuals take through “disease space.” We find that when plotting physiological parameters against each other, many pairs have hysteretic relationships that identify the current location of the host and predict the future route of the infection. These maps can readily be constructed from experimental longitudinal data, and we provide two methods to generate the maps from the cross-sectional data that is commonly gathered in field trials. We hypothesize that resilient hosts tend to take small loops through disease space, whereas nonresilient individuals take large loops. We support this hypothesis with experimental data in mice infected with Plasmodium chabaudi, finding that dying mice trace a large arc in red blood cells (RBCs) by reticulocyte space as compared to surviving mice. We find that human malaria patients who are heterozygous for sickle cell hemoglobin occupy a small area of RBCs by reticulocyte space, suggesting this approach can be used to distinguish resilience in human populations. This technique should be broadly useful in describing the in-host dynamics of infections in both model hosts and patients at both population and individual levels.

Pathologic infections are accompanied by a collection of short-term behavioral perturbations collectively termed sickness behaviors [1, 2]. These include changes in body temperature, reduced eating and drinking, and lethargy and mimic behaviors of animals in torpor and hibernation [1, 3-6]. Sickness behaviors are important, pathogen-specific components of the host response to infection [1, 3, 7-9]. In particular, host anorexia has been shown to be beneficial or detrimental depending on the infection [7, 8]. While these studies have illuminated the effects of anorexia on infection, they consider this behavior in isolation from other behaviors and from its effects on host metabolism and energy. Here, we explored the temporal dynamics of multiple sickness behaviors and their effect on host energy and metabolism throughout infection. We used the Plasmodium chabaudi AJ murine model of malaria as it causes severe pathology from which most animals recover. We found that infected animals did become anorexic, skewing their metabolism toward fatty acid oxidation and ketosis. Metabolism of fats requires oxygen for the production of ATP. In this model, animals also suffer severe anemia, limiting their ability to carry oxygen concurrent with their switch toward fatty acid metabolism. We reasoned that the combination of anorexia and anemia would increase pressure on glycolysis as a critical energy pathway because it does not require oxygen. Treating infected mice when anorexic with the glycolytic inhibitor 2-deoxyglucose (2DG) reduced survival; treating animals with glucose improved survival. Peak parasite loads were unchanged, demonstrating changes in disease tolerance. Parasite clearance was reduced with 2DG treatment, suggesting altered resistance.

Circadian rhythms enable organisms to synchronise the processes underpinning survival and reproduction to anticipate daily changes in the external environment. Recent work shows that daily (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions with their hosts. Because parasite rhythms matter for their fitness, understanding how they are regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we examine how host circadian rhythms influence rhythms in the asexual replication of malaria parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the host’s peripheral oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative to the rhythms of parasites in night-fed hosts. Our results reveal that the host’s peripheral rhythms (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, we show that parasite rhythms resynchronise to the altered host feeding rhythms when food availability is shifted, which is not mediated through rhythms in the host immune system. Our observations suggest that parasites actively control their developmental rhythms. Finally, counter to expectation, the severity of disease symptoms expressed by hosts was not affected by desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease ecology and chronobiology opens up a new arena for studying host-parasite-vector coevolution and has broad implications for applied bioscience.

17Circadian rhythms enable organisms to synchronise the processes underpinning survival and 18reproduction to anticipate daily changes in the external environment. Recent work shows that daily 19 (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions 20 with their hosts. Because parasite rhythms matter for their fitness, understanding how they are 21 regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we 22 examine how host circadian rhythms influence rhythms in the asexual replication of malaria 23 parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the 24disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of 25 hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the host's peripheral 26 oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We 27 demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative 28 to the rhythms of parasites in night-fed hosts. Our results reveal that the host's peripheral rhythms 29 (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-30 entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further 31 investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, 32 we show that parasite rhythms resynchronise to the altered host feeding rhythms when food 33 availability is shifted, which is not mediated through rhythms in the host immune system. Our 34 observations suggest that parasites actively control their developmental rhythms. Finally, counter to 35 expectation, the severity of disease symptoms expressed by hosts was not affected by 36 desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease 37 ecology and chronobiology opens up a new arena for studying host-parasite-vector coevolution and 38 has broad implications for applied bioscience. The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/229674 doi: bioRxiv preprint first posted online Dec. 6, 2017; 3 Author summary 43 How cycles of asexual replication by malaria parasites are coordinated to occur in synchrony 44 with the circadian rhythms of the host is a long-standing mystery. We reveal that rhythms associated 45 with the time-of-day that hosts feed are responsible for the timing of rhythms in parasite 46 development. Specifically, we altered host feeding time to phase-shift peripheral rhythms, whilst 47 leaving rhythms driven by the central circadian oscillator in the brain unchanged. We found that 48 parasite developmental rhythms remained synchronous but changed their phase, by 12 hours, to 49 follow the timing of host feeding. Furthermore, our results suggest that parasites themselves 50 schedule rhythms in their replication to ...