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.

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.

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.

Adaptive plasticity in the gametocyte conversion rate of malaria parasites.

Sexually reproducing parasites, such as malaria parasites, experience a trade-off between the allocation of resources to asexual replication and the production of sexual forms. Allocation by malaria parasites to sexual forms (the conversion rate) is variable but the evolutionary drivers of this plasticity are poorly understood. We use evolutionary theory for life histories to combine a mathematical model and experiments to reveal that parasites adjust conversion rate according to the dynamics of asexual densities in the blood of the host. Our model predicts the direction of change in conversion rates that returns the greatest fitness after perturbation of asexual densities by different doses of antimalarial drugs. The loss of a high proportion of asexuals is predicted to elicit increased conversion (terminal investment), while smaller losses are managed by reducing conversion (reproductive restraint) to facilitate within-host survival and future transmission. This non-linear pattern of allocation is consistent with adaptive reproductive strategies observed in multicellular organisms. We then empirically estimate conversion rates of the rodent malaria parasite Plasmodium chabaudi in response to the killing of asexual stages by different doses of antimalarial drugs and forecast the short-term fitness consequences of these responses. Our data reveal the predicted non-linear pattern, and this is further supported by analyses of previous experiments that perturb asexual stage densities using drugs or within-host competition, across multiple parasite genotypes. Whilst conversion rates, across all datasets, are most strongly influenced by changes in asexual density, parasites also modulate conversion according to the availability of red blood cell resources. In summary, increasing conversion maximises short-term transmission and reducing conversion facilitates in-host survival and thus, future transmission. Understanding patterns of parasite allocation to reproduction matters because within-host replication is responsible for disease symptoms and between-host transmission determines disease spread.

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 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.

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.

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.

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 fourfold when mosquitoes feed during their rest phase. Thus, changes in mosquito biting time (owing to bed nets) may render gametocytes less infective, but this is compensated for by the greater mosquito susceptibility.

Phenotypic plasticity in reproductive effort: malaria parasites respond to resource availability.

The trade-off between survival and reproduction is fundamental in the life history of all sexually reproducing organisms. This includes malaria parasites, which rely on asexually replicating stages for within-host survival and on sexually reproducing stages (gametocytes) for between-host transmission. The proportion of asexual stages that form gametocytes (reproductive effort) varies during infections-i.e. is phenotypically plastic-in response to changes in a number of within-host factors, including anaemia. However, how the density and age structure of red blood cell (RBC) resources shape plasticity in reproductive effort and impacts upon parasite fitness is controversial. Here, we examine how and why the rodent malaria parasite Plasmodium chabaudi alters its reproductive effort in response to experimental perturbations of the density and age structure of RBCs. We show that all four of the genotypes studied increase reproductive effort when the proportion of RBCs that are immature is elevated during host anaemia, and that the responses of the genotypes differ. We propose that anaemia (counterintuitively) generates a resource-rich environment in which parasites can afford to allocate more energy to reproduction (i.e. transmission) and that anaemia also exposes genetic variation to selection. From an applied perspective, adaptive plasticity in parasite reproductive effort could explain the maintenance of genetic variation for virulence and why anaemia is often observed as a risk factor for transmission in human infections.

Facilitation through altered resource availability in a mixed-species rodent malaria infection.

A major challenge in disease ecology is to understand how co-infecting parasite species interact. We manipulate in vivo resources and immunity to explain interactions between two rodent malaria parasites, Plasmodium chabaudi and P. yoelii. These species have analogous resource-use strategies to the human parasites Plasmodium falciparum and P. vivax: P. chabaudi and P. falciparum infect red blood cells (RBC) of all ages (RBC generalist); P. yoelii and P. vivax preferentially infect young RBCs (RBC specialist). We find that: (1) recent infection with the RBC generalist facilitates the RBC specialist (P. yoelii density is enhanced ~10 fold). This occurs because the RBC generalist increases availability of the RBC specialist's preferred resource; (2) co-infections with the RBC generalist and RBC specialist are highly virulent; (3) and the presence of an RBC generalist in a host population can increase the prevalence of an RBC specialist. Thus, we show that resources shape how parasite species interact and have epidemiological consequences.

Ecological influences on the behaviour and fertility of malaria parasites.

BACKGROUND: Sexual reproduction in the mosquito is essential for the transmission of malaria parasites and a major target for transmission-blocking interventions. Male gametes need to locate and fertilize females in the challenging environment of the mosquito blood meal, but remarkably little is known about the ecology and behaviour of male gametes. METHODS: Here, a series of experiments explores how some aspects of the chemical and physical environment experienced during mating impacts upon the production, motility, and fertility of male gametes. RESULTS AND CONCLUSIONS: Specifically, the data confirm that: (a) rates of male gametogenesis vary when induced by the family of compounds (tryptophan metabolites) thought to trigger gamete differentiation in nature; and (b) complex relationships between gametogenesis and mating success exist across parasite species. In addition, the data reveal that (c) microparticles of the same size as red blood cells negatively affect mating success; and (d) instead of swimming in random directions, male gametes may be attracted by female gametes. Understanding the mating ecology of malaria parasites, may offer novel approaches for blocking transmission and explain adaptation to different species of mosquito vectors.

The role of models in translating within-host dynamics to parasite evolution.

Mathematical modelling provides an effective way to challenge conventional wisdom about parasite evolution and investigate why parasites 'do what they do' within the host. Models can reveal when intuition cannot explain observed patterns, when more complicated biology must be considered, and when experimental and statistical methods are likely to mislead. We describe how models of within-host infection dynamics can refine experimental design, and focus on the case study of malaria to highlight how integration between models and data can guide understanding of parasite fitness in three areas: (1) the adaptive significance of chronic infections; (2) the potential for tradeoffs between virulence and transmission; and (3) the implications of within-vector dynamics. We emphasize that models are often useful when they highlight unexpected patterns in parasite evolution, revealing instead why intuition yields the wrong answer and what combination of theory and data are needed to advance understanding.

High-speed holographic microscopy of malaria parasites reveals ambidextrous flagellar waveforms.

Axonemes form the core of eukaryotic flagella and cilia, performing tasks ranging from transporting fluid in developing embryos to the propulsion of sperm. Despite their abundance across the eukaryotic domain, the mechanisms that regulate the beating action of axonemes remain unknown. The flagellar waveforms are 3D in general, but current understanding of how axoneme components interact stems from 2D data; comprehensive measurements of flagellar shape are beyond conventional microscopy. Moreover, current flagellar model systems (e.g., sea urchin, human sperm) contain accessory structures that impose mechanical constraints on movement, obscuring the "native" axoneme behavior. We address both problems by developing a high-speed holographic imaging scheme and applying it to the (male) microgametes of malaria (Plasmodium) parasites. These isolated flagella are a unique, mathematically tractable model system for the physics of microswimmers. We reveal the 3D flagellar waveforms of these microorganisms and map the differential shear between microtubules in their axonemes. Furthermore, we overturn claims that chirality in the structure of the axoneme governs the beat pattern [Hirokawa N, et al. (2009) Ann Rev Fluid Mech 41:53-72], because microgametes display a left- or right-handed character on alternate beats. This breaks the link between structural chirality in the axoneme and larger scale symmetry breaking (e.g., in developing embryos), leading us to conclude that accessory structures play a critical role in shaping the flagellar beat.

Hybridization and pre-zygotic reproductive barriers in Plasmodium.

Sexual reproduction is an obligate step in the life cycle of many parasites, including the causative agents of malaria (Plasmodium). Mixed-species infections are common in nature and consequently, interactions between heterospecific gametes occur. Given the importance of managing gene flow across parasite populations, remarkably little is understood about how reproductive isolation between species is maintained. We use the rodent malaria parasites P. berghei and P. yoelii to investigate the ecology of mixed-species mating groups, identify proteins involved in pre-zygotic barriers, and examine their evolution. Specifically, we show that (i) hybridization occurs, but at low frequency; (ii) hybridization reaches high levels when female gametes lack the surface proteins P230 or P48/45, demonstrating that these proteins are key for pre-zygotic reproductive isolation; (iii) asymmetric reproductive interference occurs, where the fertility of P. berghei gametes is reduced in the presence of P. yoelii and (iv) as expected for gamete recognition proteins, strong positive selection acts on a region of P230 and P47 (P48/45 paralogue). P230 and P48/45 are leading candidates for interventions to block malaria transmission. Our results suggest that depending on the viability of hybrids, applying such interventions to populations where mixed-species infections occur could either facilitate or hinder malaria control.

The problem of auto-correlation in parasitology.

Explaining the contribution of host and pathogen factors in driving infection dynamics is a major ambition in parasitology. There is increasing recognition that analyses based on single summary measures of an infection (e.g., peak parasitaemia) do not adequately capture infection dynamics and so, the appropriate use of statistical techniques to analyse dynamics is necessary to understand infections and, ultimately, control parasites. However, the complexities of within-host environments mean that tracking and analysing pathogen dynamics within infections and among hosts poses considerable statistical challenges. Simple statistical models make assumptions that will rarely be satisfied in data collected on host and parasite parameters. In particular, model residuals (unexplained variance in the data) should not be correlated in time or space. Here we demonstrate how failure to account for such correlations can result in incorrect biological inference from statistical analysis. We then show how mixed effects models can be used as a powerful tool to analyse such repeated measures data in the hope that this will encourage better statistical practices in parasitology.

Information use and plasticity in the reproductive decisions of malaria parasites.

BACKGROUND: Investment in the production of transmissible stages (gametocytes) and their sex ratio are malaria parasite traits that underpin mosquito infectivity and are therefore central to epidemiology. Malaria parasites adjust their levels of investment into gametocytes and sex ratio in response to changes in the in-host environment (including red blood cell resource availability, host immune responses, competition from con-specific genotypes in mixed infections, and drug treatment). This plasticity appears to be adaptive (strategic) because parasites prioritize investment (in sexual versus asexual stages and male versus female stages) in manners predicted to maximize fitness. However, the information, or 'cues' that parasites use to detect environmental changes and make appropriate decisions about investment into gametocytes and their sex ratio are unknown. METHODS: Single genotype Plasmodium chabaudi infections were exposed to 'cue' treatments consisting of intact or lysed uninfected red blood cells, lysed parasitized RBCs of the same clone or an unrelated clone, and an unmanipulated control. Infection dynamics (proportion of reticulocytes, red blood cell and asexual stage parasite densities) were monitored, and changes in gametocyte investment and sex ratio in response to cue treatments, applied either pre- or post-peak of infection were examined. RESULTS AND CONCLUSIONS: A significant reduction in gametocyte density was observed in response to the presence of lysed parasite material and a borderline significant increase in sex ratio (proportion of male gametocytes) upon exposure to lysed red blood cells (both uninfected and infected) was observed. Furthermore, the changes in gametocyte density and sex ratio in response to these cues depend on the age of infection. Demonstrating that variation in gametocyte investment and sex ratio observed during infections are a result of parasite strategies (rather than the footprint of host physiology), provides a foundation to investigate the fitness consequences of plasticity and explore whether drugs could be developed to trick parasites into making suboptimal decisions.

Sex ratio adjustment and kin discrimination in malaria parasites.

Malaria parasites and related Apicomplexans are the causative agents of the some of the most serious infectious diseases of humans, companion animals, livestock and wildlife. These parasites must undergo sexual reproduction to transmit from vertebrate hosts to vectors, and their sex ratios are consistently female-biased. Sex allocation theory, a cornerstone of evolutionary biology, is remarkably successful at explaining female-biased sex ratios in multicellular taxa, but has proved controversial when applied to malaria parasites. Here we show that, as predicted by theory, sex ratio is an important fitness-determining trait and Plasmodium chabaudi parasites adjust their sex allocation in response to the presence of unrelated conspecifics. This suggests that P. chabaudi parasites use kin discrimination to evaluate the genetic diversity of their infections, and they adjust their behaviour in response to environmental cues. Malaria parasites provide a novel way to test evolutionary theory, and support the generality and power of a darwinian approach.

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.

Testing the evolutionary drivers of malaria parasite rhythms and their consequences for host-parasite interactions.

Undertaking certain activities at the time of day that maximises fitness is assumed to explain the evolution of circadian clocks. Organisms often use daily environmental cues such as light and food availability to set the timing of their clocks. These cues may be the environmental rhythms that ultimately determine fitness, act as proxies for the timing of less tractable ultimate drivers, or are used simply to maintain internal synchrony. While many pathogens/parasites undertake rhythmic activities, both the proximate and ultimate drivers of their rhythms are poorly understood. Explaining the roles of rhythms in infections offers avenues for novel interventions to interfere with parasite fitness and reduce the severity and spread of disease. Here, we perturb several rhythms in the hosts of malaria parasites to investigate why parasites align their rhythmic replication to the host's feeding-fasting rhythm. We manipulated host rhythms governed by light, food or both, and assessed the fitness implications for parasites, and the consequences for hosts, to test which host rhythms represent ultimate drivers of the parasite's rhythm. We found that alignment with the host's light-driven rhythms did not affect parasite fitness metrics. In contrast, aligning with the timing of feeding-fasting rhythms may be beneficial for the parasite, but only when the host possess a functional canonical circadian clock. Because parasites in clock-disrupted hosts align with the host's feeding-fasting rhythms and yet derive no apparent benefit, our results suggest cue(s) from host food act as a proxy rather than being a key selective driver of the parasite's rhythm. Alternatively, parasite rhythmicity may only be beneficial because it promotes synchrony between parasite cells and/or allows parasites to align to the biting rhythms of vectors. Our results also suggest that interventions can disrupt parasite rhythms by targeting the proxies or the selective factors driving them without impacting host health.

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.