Virulence is associated with daily rhythms in the within-host replication of the malaria parasite Plasmodium chabaudi.

Most malaria (Plasmodium spp.) parasite species undergo asexual replication synchronously within the red blood cells of their vertebrate host. Rhythmicity in this intraerythrocytic developmental cycle (IDC) enables parasites to maximise exploitation of the host and align transmission activities with the time of day that mosquito vectors blood feed. The IDC is also responsible for the major pathologies associated with malaria, and plasticity in the parasite's rhythm can confer tolerance to antimalarial drugs. Both the severity of infection (virulence) and synchrony of the IDC vary across species and between genotypes of Plasmodium; however, this variation is poorly understood. The theory predicts that virulence and IDC synchrony are negatively correlated, and we tested this hypothesis using two closely related genotypes of the rodent malaria model Plasmodium chabaudi that differ markedly in virulence. We also test the predictions that, in response to perturbations to the timing (phase) of the IDC schedule relative to the phase of host rhythms (misalignment), the virulent parasite genotype recovers the correct phase relationship faster, incurs less fitness losses and so hosts benefit less from misalignment when infected with a virulent genotype. Our predictions are partially supported by results suggesting that the virulent parasite genotype is less synchronous in some circumstances and recovers faster from misalignment. While hosts were less anaemic when infected by misaligned parasites, the extent of this benefit did not depend on parasite virulence. Overall, our results suggest that interventions to perturb the alignment between the IDC schedule, and host rhythms and increase synchrony between parasites within each IDC, could alleviate disease symptoms. However, virulent parasites, which are better at withstanding conventional antimalarial treatment, would also be intrinsically better able to tolerate such interventions.

Machine learning reveals singing rhythms of male Pacific field crickets are clock controlled.

Circadian rhythms are ubiquitous in nature and endogenous circadian clocks drive the daily expression of many fitness-related behaviors. However, little is known about whether such traits are targets of selection imposed by natural enemies. In Hawaiian populations of the nocturnally active Pacific field cricket (Teleogryllus oceanicus), males sing to attract mates, yet sexually selected singing rhythms are also subject to natural selection from the acoustically orienting and deadly parasitoid fly, Ormia ochracea. Here, we use T. oceanicus to test whether singing rhythms are endogenous and scheduled by circadian clocks, making them possible targets of selection imposed by flies. We also develop a novel audio-to-circadian analysis pipeline, capable of extracting useful parameters from which to train machine learning algorithms and process large quantities of audio data. Singing rhythms fulfilled all criteria for endogenous circadian clock control, including being driven by photoschedule, self-sustained periodicity of approximately 24 h, and being robust to variation in temperature. Furthermore, singing rhythms varied across individuals, which might suggest genetic variation on which natural and sexual selection pressures can act. Sexual signals and ornaments are well-known targets of selection by natural enemies, but our findings indicate that the circadian timing of those traits' expression may also determine fitness.

Testing a non-destructive assay to track Plasmodium sporozoites in mosquitoes over time.

BACKGROUND: The extrinsic incubation period (EIP), defined as the time it takes for malaria parasites in a mosquito to become infectious to a vertebrate host, is one of the most influential parameters for malaria transmission but remains poorly understood. The EIP is usually estimated by quantifying salivary gland sporozoites in subsets of mosquitoes, which requires terminal sampling. However, assays that allow repeated sampling of individual mosquitoes over time could provide better resolution of the EIP. METHODS: We tested a non-destructive assay to quantify sporozoites of two rodent malaria species, Plasmodium chabaudi and Plasmodium berghei, expelled throughout 24-h windows, from sugar-soaked feeding substrates using quantitative-PCR. RESULTS: The assay is able to quantify sporozoites from sugar-soaked feeding substrates, but the prevalence of parasite-positive substrates was low. Various methods were attempted to increase the detection of expelled parasites (e.g. running additional technical replicates; using groups rather than individual mosquitoes), but these did not increase the detection rate, suggesting that expulsion of sporozoites is variable and infrequent. CONCLUSIONS: We reveal successful detection of expelled sporozoites from sugar-soaked feeding substrates. However, investigations of the biological causes underlying the low detection rate of sporozoites (e.g. mosquito feeding behaviour, frequency of sporozoite expulsion or sporozoite clumping) are needed to maximise the utility of using non-destructive assays to quantify sporozoite dynamics. Increasing detection rates will facilitate the detailed investigation on infection dynamics within mosquitoes, which is necessary to explain the highly variable EIP of Plasmodium and to improve understanding of malaria transmission dynamics.

The impact of innate immunity on malaria parasite infection dynamics in rodent models.

Decades of research have probed the molecular and cellular mechanisms that control the immune response to malaria. Yet many studies offer conflicting results on the functional impact of innate immunity for controlling parasite replication early in infection. We conduct a meta-analysis to seek consensus on the effect of innate immunity on parasite replication, examining three different species of rodent malaria parasite. Screening published studies that span four decades of research we collate, curate, and statistically analyze infection dynamics in immune-deficient or -augmented mice to identify and quantify general trends and reveal sources of disagreement among studies. Additionally, we estimate whether host factors or experimental methodology shape the impact of immune perturbations on parasite burden. First, we detected meta-analytic mean effect sizes (absolute Cohen's h) for the difference in parasite burden between treatment and control groups ranging from 0.1475 to 0.2321 across parasite species. This range is considered a small effect size and translates to a modest change in parasitaemia of roughly 7-12% on average at the peak of infection. Second, we reveal that variation across studies using P. chabaudi or P. yoelii is best explained by stochasticity (due to small sample sizes) rather than by host factors or experimental design. Third, we find that for P. berghei the impact of immune perturbation is increased when young or female mice are used and is greatest when effector molecules (as opposed to upstream signalling molecules) are disrupted (up to an 18% difference in peak parasitaemia). Finally, we find little evidence of publication bias suggesting that our results are robust. The small effect sizes we observe, across three parasite species, following experimental perturbations of the innate immune system may be explained by redundancy in a complex biological system or by incomplete (or inappropriate) data reporting for meta-analysis. Alternatively, our findings might indicate a need to re-evaluate the efficiency with which innate immunity controls parasite replication early in infection. Testing these hypotheses is necessary to translate understanding from model systems to human malaria.

Circadian rhythmicity in murine blood: Electrical effects of malaria infection and anemia.

Circadian rhythms are biological adaptations to the day-night cycle, whereby cells adapt to changes in the external environment or internal physiology according to the time of day. Whilst many cellular clock mechanisms involve gene expression feedback mechanisms, clocks operate even where gene expression is absent. For example, red blood cells (RBCs) do not have capacity for gene expression, and instead possess an electrophysiological oscillator where cytosolic potassium plays a key role in timekeeping. We examined murine blood under normal conditions as well as in two perturbed states, malaria infection and induced anemia, to assess changes in baseline cellular electrophysiology and its implications for the electrophysiological oscillator. Blood samples were analyzed at 4-h intervals over 2 days by dielectrophoresis, and microscopic determination of parasitemia. We found that cytoplasmic conductivity (indicating the concentration of free ions in the cytoplasm and related to the membrane potential) exhibited circadian rhythmic behavior in all three cases (control, malaria and anemia). Compared to control samples, cytoplasm conductivity was decreased in the anemia group, whilst malaria-infected samples were in antiphase to control. Furthermore, we identified rhythmic behavior in membrane capacitance of malaria infected cells that was not replicated in the other samples. Finally, we reveal the historically famous rhythmicity of malaria parasite replication is in phase with cytoplasm conductivity. Our findings suggest the electrophysiological oscillator can impact on malaria parasite replication and/or is vulnerable to perturbation by rhythmic parasite activities.

Targeting malaria parasites inside mosquitoes: ecoevolutionary consequences.

Proof-of-concept studies demonstrate that antimalarial drugs designed for human treatment can also be applied to mosquitoes to interrupt malaria transmission. Deploying a new control tool is ideally undertaken within a stewardship programme that maximises a drug's lifespan by minimising the risk of resistance evolution and slowing its spread once emerged. We ask: what are the epidemiological and evolutionary consequences of targeting parasites within mosquitoes? Our synthesis argues that targeting parasites inside mosquitoes (i) can be modelled by readily expanding existing epidemiological frameworks; (ii) provides a functionally novel control method that has potential to be more robust to resistance evolution than targeting parasites in humans; and (iii) could extend the lifespan and clinical benefit of antimalarials used exclusively to treat humans.

Vector control: agents of selection on malaria parasites?

Insect vectors are responsible for spreading many infectious diseases, yet interactions between pathogens/parasites and insect vectors remain poorly understood. Filling this knowledge gap matters because vectors are evolving in response to the deployment of vector control tools (VCTs). Yet, whilst the evolutionary responses of vectors to VCTs are being carefully monitored, the knock-on consequences for parasite evolution have been overlooked. By examining how mosquito responses to VCTs impact upon malaria parasite ecology, we derive a framework for predicting parasite responses. Understanding how VCTs affect the selection pressures imposed on parasites could help to mitigate against parasite evolution that leads to unfavourable epidemiological outcomes. Furthermore, anticipating parasite evolution will inform monitoring strategies for VCT programmes as well as uncovering novel VCT strategies.

Mistimed malaria parasites re-synchronize with host feeding-fasting rhythms by shortening the duration of intra-erythrocytic development.

AIMS: Malaria parasites exhibit daily rhythms in the intra-erythrocytic development cycle (IDC) that underpins asexual replication in the blood. The IDC schedule is aligned with the timing of host feeding-fasting rhythms. When the IDC schedule is perturbed to become mismatched to host rhythms, it readily reschedules but it is not known how. METHODS: We intensively follow four groups of infections that have different temporal alignments between host rhythms and the IDC schedule for 10 days, before and after the peak in asexual densities. We compare how the duration, synchrony and timing of the IDC differs between parasites in control infections and those forced to reschedule by 12 hours and ask whether the density of parasites affects the rescheduling process. RESULTS AND CONCLUSIONS: Our experiments reveal parasites shorten the IDC duration by 2-3 hours to become realigned to host feeding-fasting rhythms with 5-6 days, in a density-independent manner. Furthermore, parasites are able to reschedule without significant fitness costs for them or their hosts. Understanding the extent of, and limits on, plasticity in the IDC schedule may reveal targets for novel interventions, such as drugs to disrupt IDC regulation and preventing IDC dormancy conferring tolerance to existing drugs.

Synchrony between daily rhythms of malaria parasites and hosts is driven by an essential amino acid.

Background: Rapid asexual replication of blood stage malaria parasites is responsible for the severity of disease symptoms and fuels the production of transmission forms. Here, we demonstrate that the Plasmodium chabaudi's schedule for asexual replication can be orchestrated by isoleucine, a metabolite provided to the parasite in periodic manner due to the host's rhythmic intake of food. Methods: We infect female C57BL/6 and Per1/2-null TTFL clock-disrupted mice with 1×10 5 red blood cells containing P. chabaudi (DK genotype). We perturb the timing of rhythms in asexual replication and host feeding-fasting cycles to identify nutrients with rhythms that match all combinations of host and parasite rhythms. We then test whether perturbing the availability of the best candidate nutrient in vitro elicits changes their schedule for asexual development. Results: Our large-scale metabolomics experiment and follow up experiments reveal that only one metabolite - the amino acid isoleucine - fits criteria for a time-of-day cue used by parasites to set the schedule for replication. The response to isoleucine is a parasite strategy rather than solely the consequences of a constraint imposed by host rhythms, because unlike when parasites are deprived of other essential nutrients, they suffer no apparent costs from isoleucine withdrawal. Conclusions: Overall, our data suggest parasites can use the daily rhythmicity of blood-isoleucine concentration to synchronise asexual development with the availability of isoleucine, and potentially other resources, that arrive in the blood in a periodic manner due to the host's daily feeding-fasting cycle. Identifying both how and why parasites keep time opens avenues for interventions; interfering with the parasite's time-keeping mechanism may stall replication, increasing the efficacy of drugs and immune responses, and could also prevent parasites from entering dormancy to tolerate drugs.

Daily rhythms of both host and parasite affect antimalarial drug efficacy.

BACKGROUND AND OBJECTIVES: Circadian rhythms contribute to treatment efficacy in several non-communicable diseases. However, chronotherapy (administering drugs at a particular time-of-day) against infectious diseases has been overlooked. Yet, the daily rhythms of both hosts and disease-causing agents can impact the efficacy of drug treatment. We use the rodent malaria parasite Plasmodium chabaudi, to test whether the daily rhythms of hosts, parasites and their interactions affect sensitivity to the key antimalarial, artemisinin. METHODOLOGY: Asexual malaria parasites develop rhythmically in the host's blood, in a manner timed to coordinate with host daily rhythms. Our experiments coupled or decoupled the timing of parasite and host rhythms, and we administered artemisinin at different times of day to coincide with when parasites were either at an early (ring) or later (trophozoite) developmental stage. We quantified the impacts of parasite developmental stage, and alignment of parasite and host rhythms, on drug sensitivity. RESULTS: We find that rings were less sensitive to artemisinin than trophozoites, and this difference was exacerbated when parasite and host rhythms were misaligned, with little direct contribution of host time-of-day on its own. Furthermore, the blood concentration of haem at the point of treatment correlated positively with artemisinin efficacy but only when parasite and host rhythms were aligned. CONCLUSIONS AND IMPLICATIONS: Parasite rhythms influence drug sensitivity in vivo. The hitherto unknown modulation by alignment between parasite and host daily rhythms suggests that disrupting the timing of parasite development could be a novel chronotherapeutic approach. LAY SUMMARY: We reveal that chronotherapy (providing medicines at a particular time-of-day) could improve treatment for malaria infections. Specifically, parasites' developmental stage at the time of treatment and the coordination of timing between parasite and host both affect how well antimalarial drug treatment works.

The private life of malaria parasites: Strategies for sexual reproduction.

Malaria parasites exhibit a complex lifecycle, requiring extensive asexual replication in the liver and blood of the vertebrate host, and in the haemocoel of the insect vector. Yet, they must also undergo a single round of sexual reproduction, which occurs in the vector's midgut upon uptake of a blood meal. Sexual reproduction is obligate for infection of the vector and thus, is essential for onwards transmission to new hosts. Sex in malaria parasites involves several bottlenecks in parasite number, making the stages involved attractive targets for blocking disease transmission. Malaria parasites have evolved a suite of adaptations ("strategies") to maximise the success of sexual reproduction and transmission, which could undermine transmission-blocking interventions. Yet, understanding parasite strategies may also reveal novel opportunities for such interventions. Here, we outline how evolutionary and ecological theories, developed to explain reproductive strategies in multicellular taxa, can be applied to explain two reproductive strategies (conversion rate and sex ratio) expressed by malaria parasites within the vertebrate host.

Ecology of asynchronous asexual replication: the intraerythrocytic development cycle of Plasmodium berghei is resistant to host rhythms.

BACKGROUND: Daily periodicity in the diverse activities of parasites occurs across a broad taxonomic range. The rhythms exhibited by parasites are thought to be adaptations that allow parasites to cope with, or exploit, the consequences of host activities that follow daily rhythms. Malaria parasites (Plasmodium) are well-known for their synchronized cycles of replication within host red blood cells. Whilst most species of Plasmodium appear sensitive to the timing of the daily rhythms of hosts, and even vectors, some species present no detectable rhythms in blood-stage replication. Why the intraerythrocytic development cycle (IDC) of, for example Plasmodium chabaudi, is governed by host rhythms, yet seems completely independent of host rhythms in Plasmodium berghei, another rodent malaria species, is mysterious. METHODS: This study reports a series of five experiments probing the relationships between the asynchronous IDC schedule of P. berghei and the rhythms of hosts and vectors by manipulating host time-of-day, photoperiod and feeding rhythms. RESULTS: The results reveal that: (i) a lack coordination between host and parasite rhythms does not impose appreciable fitness costs on P. berghei; (ii) the IDC schedule of P. berghei is impervious to host rhythms, including altered photoperiod and host-feeding-related rhythms; (iii) there is weak evidence for daily rhythms in the density and activities of transmission stages; but (iv), these rhythms have little consequence for successful transmission to mosquitoes. CONCLUSIONS: Overall, host rhythms do not affect the performance of P. berghei and its asynchronous IDC is resistant to the scheduling forces that underpin synchronous replication in closely related parasites. This suggests that natural variation in the IDC schedule across species represents different parasite strategies that maximize fitness. Thus, subtle differences in the ecological interactions between parasites and their hosts/vectors may select for the evolution of very different IDC schedules.

Automated detection and staging of malaria parasites from cytological smears using convolutional neural networks.

Microscopic examination of blood smears remains the gold standard for laboratory inspection and diagnosis of malaria. Smear inspection is, however, time-consuming and dependent on trained microscopists with results varying in accuracy. We sought to develop an automated image analysis method to improve accuracy and standardization of smear inspection that retains capacity for expert confirmation and image archiving. Here, we present a machine learning method that achieves red blood cell (RBC) detection, differentiation between infected/uninfected cells, and parasite life stage categorization from unprocessed, heterogeneous smear images. Based on a pretrained Faster Region-Based Convolutional Neural Networks (R-CNN) model for RBC detection, our model performs accurately, with an average precision of 0.99 at an intersection-over-union threshold of 0.5. Application of a residual neural network-50 model to infected cells also performs accurately, with an area under the receiver operating characteristic curve of 0.98. Finally, combining our method with a regression model successfully recapitulates intraerythrocytic developmental cycle with accurate lifecycle stage categorization. Combined with a mobile-friendly web-based interface, called PlasmoCount, our method permits rapid navigation through and review of results for quality assurance. By standardizing assessment of Giemsa smears, our method markedly improves inspection reproducibility and presents a realistic route to both routine lab and future field-based automated malaria diagnosis.

Host circadian clocks do not set the schedule for the within-host replication of malaria parasites.

Circadian clocks coordinate organisms' activities with daily cycles in their environment. Parasites are subject to daily rhythms in the within-host environment, resulting from clock-control of host activities, including immune responses. Parasites also exhibit rhythms in their activities: the timing of within-host replication by malaria parasites is coordinated to host feeding rhythms. Precisely which host feeding-related rhythm(s) parasites align with and how this is achieved are unknown. Understanding rhythmic replication in malaria parasites matters because it underpins disease symptoms and fuels transmission investment. We test if rhythmicity in parasite replication is coordinated with the host's feeding-related rhythms and/or rhythms driven by the host's canonical circadian clock. We find that parasite rhythms coordinate with the time of day that hosts feed in both wild-type and clock-mutant hosts, whereas parasite rhythms become dampened in clock-mutant hosts that eat continuously. Our results hold whether infections are initiated with synchronous or with desynchronized parasites. We conclude that malaria parasite replication is coordinated to rhythmic host processes that are independent of the core-clock proteins PERIOD 1 and 2; most likely, a periodic nutrient made available when the host digests food. Thus, novel interventions could disrupt parasite rhythms to reduce their fitness, without interference by host clock-controlled homeostasis.

Malaria parasites regulate intra-erythrocytic development duration via serpentine receptor 10 to coordinate with host rhythms.

Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 h suggesting a circadian basis, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches utilizing rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit daily rhythms in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 6% of Plasmodium falciparum genes show 24 h rhythms in expression under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a 24 h transcriptional rhythm and disrupting it in rodent malaria parasites shortens the IDC by 2-3 h; (v) Multiple processes including DNA replication, and the ubiquitin and proteasome pathways, are affected by loss of coordination with host rhythms and by disruption of SR10. Our results reveal malaria parasites are at least partly responsible for scheduling the IDC and coordinating their development with host daily rhythms.

Periodic Parasites and Daily Host Rhythms.

Biological rhythms appear to be an elegant solution to the challenge of coordinating activities with the consequences of the Earth's daily and seasonal rotation. The genes and molecular mechanisms underpinning circadian clocks in multicellular organisms are well understood. In contrast, the regulatory mechanisms and fitness consequences of biological rhythms exhibited by parasites remain mysterious. Here, we explore how periodicity in parasite traits is generated and why daily rhythms matter for parasite fitness. We focus on malaria (Plasmodium) parasites which exhibit developmental rhythms during replication in the mammalian host's blood and in transmission to vectors. Rhythmic in-host parasite replication is responsible for eliciting inflammatory responses, the severity of disease symptoms, and fueling transmission, as well as conferring tolerance to anti-parasite drugs. Thus, understanding both how and why the timing and synchrony of parasites are connected to the daily rhythms of hosts and vectors may make treatment more effective and less toxic to hosts.

Testing possible causes of gametocyte reduction in temporally out-of-synch malaria infections.

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.

Adaptive phenotypic plasticity in malaria parasites is not constrained by previous responses to environmental change.

BACKGROUND AND OBJECTIVES: Phenotypic plasticity enables organisms to maximize fitness by matching trait values to different environments. Such adaptive phenotypic plasticity is exhibited by parasites, which experience frequent environmental changes during their life cycle, between individual hosts and also in within-host conditions experienced during infections. Life history theory predicts that the evolution of adaptive phenotypic plasticity is limited by costs and constraints, but tests of these concepts are scarce. METHODOLOGY: Here, we induce phenotypic plasticity in malaria parasites to test whether mounting a plastic response to an environmental perturbation constrains subsequent plastic responses to further environmental change. Specifically, we perturb red blood cell resource availability to induce Plasmodium chabaudi to alter the trait values of several phenotypes underpinning within-host replication and between-host transmission. We then transfer parasites to unperturbed hosts to examine whether constraints govern the parasites' ability to alter these phenotypes in response to their new in-host environment. RESULTS: Parasites alter trait values in response to the within-host environment they are exposed to. We do not detect negative consequences, for within-host replication or between-host transmission, of previously mounting a plastic response to a perturbed within-host environment. CONCLUSIONS AND IMPLICATIONS: We suggest that malaria parasites are highly plastic and adapted to adjusting their phenotypes in response to the frequent changes in the within-host conditions they experience during infections. Our findings support the growing body of evidence that medical interventions, such as anti-parasite drugs, induce plastic responses that are adaptive and can facilitate the survival and potentially, drug resistance of parasites. LAY SUMMARY: Malaria parasites have evolved flexible strategies to cope with the changing conditions they experience during infections. We show that using such flexible strategies does not impact upon the parasites' ability to grow (resulting in disease symptoms) or transmit (spreading the disease).

Plasticity and genetic variation in traits underpinning asexual replication of the rodent malaria parasite, Plasmodium chabaudi.

BACKGROUND: The ability of malaria (Plasmodium) parasites to adjust investment into sexual transmission stages versus asexually replicating stages is well known, but plasticity in other traits underpinning the replication rate of asexual stages in the blood has received less attention. Such traits include burst size (the number of merozoites produced per schizont), the duration of the asexual cycle, and invasion preference for different ages of red blood cell (RBC). METHODS: Here, plasticity [environment (E) effects] and genetic variation [genotype (G) effects] in traits relating to asexual replication rate are examined for 4 genotypes of the rodent malaria parasite Plasmodium chabaudi. An experiment tested whether asexual dynamics differ between parasites infecting control versus anaemic hosts, and whether variation in replication rate can be explained by differences in burst size, asexual cycle, and invasion rates. RESULTS: The within-host environment affected each trait to different extents but generally had similar impacts across genotypes. The dynamics of asexual densities exhibited a genotype by environment effect (G×E), in which one of the genotypes increased replication rate more than the others in anaemic hosts. Burst size and cycle duration varied between the genotypes (G), while burst size increased and cycle duration became longer in anaemic hosts (E). Variation in invasion rates of differently aged RBCs was not explained by environmental or genetic effects. Plasticity in burst size and genotype are the only traits making significant contributions to the increase in asexual densities observed in anaemic hosts, together explaining 46.4% of the variation in replication rate. CONCLUSIONS: That host anaemia induces several species of malaria parasites to alter conversion rate is well documented. Here, previously unknown plasticity in other traits underpinning asexual replication is revealed. These findings contribute to mounting evidence that malaria parasites deploy a suite of sophisticated strategies to maximize fitness by coping with, or exploiting the opportunities provided by, the variable within-host conditions experienced during infections. That genetic variation and genotype by environment interactions also shape these traits highlights their evolutionary potential. Asexual replication rate is a major determinant of virulence and so, understanding the evolution of virulence requires knowledge of the ecological (within-host environment) and genetic drivers of variation among parasites.

Time-of-day of blood-feeding: effects on mosquito life history and malaria transmission.

BACKGROUND: Biological rhythms allow organisms to compartmentalise and coordinate behaviours, physiologies, and cellular processes with the predictable daily rhythms of their environment. There is increasing recognition that the biological rhythms of mosquitoes that vector parasites are important for global health. For example, whether perturbations in blood foraging rhythms as a consequence of vector control measures can undermine disease control. To address this, we explore the impacts of altered timing of blood-feeding on mosquito life history traits and malaria transmission. METHODS: We present three experiments in which Anopheles stephensi mosquitoes were fed in the morning or evening on blood that had different qualities, including: (i) chemical-induced or (ii) Plasmodium chabaudi infection-induced anaemia; (iii) Plasmodium berghei infection but no anaemia; or (iv) stemming from hosts at different times of day. We then compared whether time-of-day variation in blood meal characteristics influences mosquito fitness proxies relating to survival and reproduction, and malaria transmission proxies. RESULTS: Mosquito lifespan is not influenced by the time-of-day they received a blood meal, but several reproductive metrics are affected, depending on other blood characteristics. Overall, our data suggest that receiving a blood meal in the morning makes mosquitoes more likely to lay eggs, lay slightly sooner and have a larger clutch size. In keeping with previous work, P. berghei infection reduces mosquito lifespan and the likelihood of laying eggs, but time-of-day of blood-feeding does not impact upon these metrics nor on transmission of this parasite. CONCLUSION: The time-of-day of blood-feeding does not appear to have major consequences for mosquito fitness or transmission of asynchronous malaria species. If our results from a laboratory colony of mosquitoes living in benign conditions hold for wild mosquitoes, it suggests that mosquitoes have sufficient flexibility in their physiology to cope with changes in biting time induced by evading insecticide-treated bed nets. Future work should consider the impact of multiple feeding cycles and the abiotic stresses imposed by the need to forage for blood during times of day when hosts are not protected by bed nets.