Host circadian rhythms are disrupted during malaria infection in parasite genotype-specific manners

Infection can dramatically alter behavioural and physiological traits as hosts become sick and subsequently return to health. Such “sickness behaviours” include disrupted circadian rhythms in both locomotor activity and body temperature. Host sickness behaviours vary in pathogen species-specific manners but the influence of pathogen intraspecific variation is rarely studied. We examine how infection with the murine malaria parasite, Plasmodium chabaudi, shapes sickness in terms of parasite genotype-specific effects on host circadian rhythms. We reveal that circadian rhythms in host locomotor activity patterns and body temperature become differentially disrupted and in parasite genotype-specific manners. Locomotor activity and body temperature in combination provide more sensitive measures of health than commonly used virulence metrics for malaria (e.g. anaemia). Moreover, patterns of host disruption cannot be explained simply by variation in replication rate across parasite genotypes or the severity of anaemia each parasite genotype causes. It is well known that disruption to circadian rhythms is associated with non-infectious diseases, including cancer, type 2 diabetes, and obesity. Our results reveal that disruption of host circadian rhythms is a genetically variable virulence trait of pathogens with implications for host health and disease tolerance

Adaptive periodicity in the infectivity of malaria gametocytes to mosquitoes

Daily rhythms in behaviour, physiology, and molecular processes are expected to enable organisms to appropriately schedule activities according to consequences of the daily rotation of the Earth. For parasites, this includes capitalizing on periodicity in transmission opportunities and for hosts/vectors, this may select for rhythms in immune defence. We examine rhythms in the density and infectivity of transmission forms (gametocytes) of rodent malaria parasites in the host’s blood, parasite development inside mosquito vectors, and potential for onwards transmission. Furthermore, we simultaneously test whether mosquitoes exhibit rhythms in susceptibility. We reveal that at night, gametocytes are twice as infective, despite being less numerous in the blood. Enhanced infectiousness at night interacts with mosquito rhythms to increase sporozoite burdens four-fold when mosquitoes feed during their rest phase. Thus, changes in mosquito biting time (due to bed nets) may render gametocytes less infective, but this is compensated for by the greater mosquito susceptibility

Timing of host feeding drives rhythms in parasite replication

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 hosts' 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 hosts' 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

Plasmodium schizont densities in culture grown with and without isoleucine, as well as isoleucine starvation for different durations of time

To test the effect of the amino acid isoleucine on the parasite asexual cycle we compared parasite developmental progression in cultures with and without isoleucine (50 mg/L), as well as after different durations (7, 14, 18 hours) of isoleucine starvation

Plasmodium schizont proportion in culture grown with and without isoleucine

To test the effect of the amino acid isoleucine on the parasite asexual cycle we compared parasite developmental progression in cultures with and without isoleucine (50 mg/L), as well as after different durations (7, 14, 18 hours) of isoleucine starvation

Targeted metabolomics on malaria-infected mouse blood

We sampled mice in blocks (A-D), meaning each individual mouse was sampled every eight hours during the 26-hour sampling window, with 14 time points in total. We did not sample each mouse at each sampling point to minimise the total volume of blood being taken. We analysed blood plasma samples using the AbsoluteIDQ p180 targeted metabolomics kit (Biocrates Life Sciences AG, Innsbruck, Austria) and a Waters Xevo TQ-S mass spectrometer coupled to an Acquity UPLC system (Waters Corporation, Milford, MA, USA). We prepared the plasma samples (10 μl) according to the manufacturer’s instructions, adding several stable isotope–labelled standards to the samples prior to the derivatization and extraction steps. Using UPLC/MS (ultra performance liquid chromatography/mass spectrometry), we quantified 185 metabolites from 5 different compound classes (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, and sphingolipids). We ran the samples on two 96-well plates, randomised the sample order and ran three levels of quality control (QC) on each plate. We normalised the data between the plates using the results of quality control level 2 (QC2) repeats across the plate (n=4) and between plates (n=2) using Biocrates METIDQ software (QC2 correction)

Plasmodium chabaudi genotype DK parasite stages during time series

After infecting mice intravenously with 1×105 P. chabaudi DK genotype infected RBC at ring stage we made a thin blood smear on day 5 post infection and again every 2 hours to quantify parasite rhythms by counting ~100 parasites where possible per blood smear using compound microscopy. Following (Prior et al 2018, O’Donnell et al 2019 and Rijo-Ferreira et al 2020), we used the proportion of ring stages as a phase marker (an estimate of the timing of parasite development in the blood) of parasite rhythms