Petra Schneider

Synchronised multiplication of Plasmodium parasites within red blood cells causes periodic malaria fevers. Aligning blood stage development with the feeding-fasting rhythm of the vertebrate host facilitates within-host survival and between-host transmission. We use the rodent model Plasmodium chabaudi to test when, following development in the liver, the blood stage of infection begins. We find egress from the liver into the blood is aligned with the time of day of rhythmic host feeding, but only in wild type hosts, with egress occurring after a fixed period of pre-erythrocytic development in hosts without a canonical circadian clock. However, perturbing the duration over which parasites enter the bloodstream does not affect their multiplication rate in the first five intraerythrocytic development cycles, suggesting that the expected fitness benefits from timing egress anticipates rhythmic challenges or opportunities (e.g. rhythmic nutrient limitation or nutrient availability) later in the infection when parasite densities increase.

Synchronised multiplication of Plasmodium parasites within red blood cells causes periodic malaria fevers. Aligning blood-stage development with the vertebrate host’s feeding-fasting rhythm facilitates within-host survival and between-host transmission. We use the rodent model Plasmodium chabaudi to test when, following development in the liver, the blood stage of infection begins. We find egress from the liver into the blood is aligned with the time of day of rhythmic host feeding, but only in wild type hosts, with egress occurring after a fixed period of pre-erythrocytic development in hosts without a functional canonical clock. However, perturbing the duration over which parasites enter the bloodstream does not affect their multiplication rate in the first few IDCs, suggesting fitness benefits from timing egress anticipates rhythmic challenges or opportunities later in the infection.

Background: Circadian clocks are thought to have evolved owing to the benefits of anticipating daily environmental rhythms. Daily environmental rhythms that impact on fitness include interactions between organisms, such as host–pathogen interactions. For example, host susceptibility to infection for taxonomically diverse hosts and pathogens varies across the circadian cycle. We previously revealed that mosquito vectors are less susceptible to malaria (Plasmodium) infection during their active phase (night time), and here we test whether a similar pattern occurs for infection of the mammalian host. Methods: We used Plasmodium berghei-infected Anopheles stephensi mosquitoes to infect mice during their rest or active phase, both in untreated and pyrimethamine-treated mice. We assessed the parasites’ success in establishing at the first site of replication (in the liver) by quantifying parasite burdens using quantitative PCR (qPCR), adjusted for sporozoite inocula. By independently manipulating the photoschedules of vectors and hosts, we standardise the time-of-day for parasites and mosquitoes used to initiate infections, and thus, directly test the impact of host time-of-day on the parasites’ ability to establish an infection. Results: The three experiments we conducted showed that pyrimethamine treatment reduced parasite liver burdens, but not in a biologically significant manner dependent on host time-of-day (active/rest phase). Furthermore, host time-of-day did not affect parasite liver burdens in untreated hosts. Conclusions: Understanding the roles of host, parasite, and vector rhythms on malaria transmission is important given that mosquitoes are altering the time of day that they bite. That rhythms, per se, do not affect vector-to-host transmission suggests that the impacts of time-of-day on components of vectorial capacity are more epidemiologically influential than host rhythms. Graphical

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.

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 chabaudiandPlasmodium 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 ofPlasmodiumand to improve understanding of malaria transmission dynamics.Graphical

Asexually replicating stages of most malaria ( Plasmodium spp.) parasite species replicate 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 , yet this variation is poorly understood. 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 loss, and so, hosts benefit less from misalignment of the virulent genotype. Our predictions are partially supported; the virulent parasite genotype was less synchronous in some circumstances and recovered 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.

RNA extraction from 5uL mouse blood samples, for subsequent cDNA production and qPCR to detect Plasmodium parasites - specifically gametocytes - within the blood. Using KingFisher Flex (96 well) system with MagMAX-96 Total RNA Isolation Kit. Protocol to import into BindIt Software attached.

DNA extraction from 5uL mouse blood samples, for subsequent qPCR to detect Plasmodium parasites within the blood. Using KingFisher Flex (96 well) system with MagMAX™-96 DNA Multi-Sample Kit. Protocol to import into BindIt Software attached.