Biology subjects

Graumans, W.

Publications and source records attributed to Graumans, W..

8 recordsLinked to original sources

bioRxiv2026

Quantifying sporozoite inoculum dynamics with the SpitGrid reveals temporal decay and behavioral determinants of Plasmodium transmission

The number of Plasmodium sporozoites expelled by an infected mosquito during a bite, the inoculum size, is a critical but rarely measured factor in transmission. Its temporal dynamics, individual variation, and behavioral underpinnings remain poorly understood. To address this, we developed the SpitGrid, a modular platform that supports natural probing and feeding on artificial substrates, while enabling behavioral characterization combined with non-lethal, sensitive measurement of inoculum size for individual mosquitoes in high-throughput. Using the SpitGrid, we determined inoculum size of Plasmodium falciparum-infected Anopheles stephensi mosquitoes over time and identified a robust temporal structure. Inoculum size peaked shortly after the extrinsic incubation period, and declined approximately log-linearly post peak. Salivary gland parasite load decreased only moderately and was positively associated with inoculum size during the early phase of sporozoite expelling, yet explained only part of the variation observed in inoculum size. Fine-grained behavioral classification confirmed that probing alone can deliver substantial inocula, and revealed that probing more often increased inoculum size while the duration of probing, or if this led to feeding, had little effect. Application of the SpitGrid to Plasmodium vivax-infected mosquitoes similarly showed a temporally structured expelling profile, with generally smaller inocula and a higher proportion of non-expelling mosquitoes. Our observations reveal that Plasmodium infectivity in mosquitoes is a highly dynamics process, challenging the assumption that per-bite transmission potential remains constant over time. We establish the SpitGrid as a flexible tool to gain a nuanced understanding of the determinants of transmission, refine epidemiological models, and evaluate the efficacy of vector- based interventions under biologically relevant conditions. HighlightsWe develop the SpitGrid, a flexible, accessible, low-cost and animal free platform to measure sporozoite expelling We identify a peak in P. falciparum and P. vivax sporozoite expelling by infected Anopheles stephensi mosquitoes early after completion of the extrinsic incubation period, followed by a gradual decline We confirm a correlation between salivary gland load and inoculum size that weakens over time, indicating that a smaller fraction of the remaining salivary gland load is being expelled by older mosquitoes We confirm that mosquito probing alone can deliver substantial sporozoite inoculum independently of a successful bloodmeal By dissecting the behavioral parameters that determine inoculum size, we observe that the initiation of probing is a major determinant of inoculum size with relatively minor roles for probing time and whether biting results in engorgement Compared to P. falciparum, we observe comparatively smaller inocula, in P. vivax-infected mosquitoes a higher rate of non-expellers and similar albeit more moderate temporal structure with an earlier onset of expelling, illustrating cross-species utility of the SpitGrid

microbiology↗

bioRxiv2025

Expelling of P. falciparum sporozoites by Anopheles stephensi mosquitoes during repeated feeding

It is currently unclear whether repeated mosquito feeding attempts achieve pathogen transmission. We measured Plasmodium falciparum sporozoite expelling in Anopheles stephensi mosquitoes during two interrupted feeding attempts. The number of expelled sporozoites was positively correlated with salivary gland sporozoite load during the first feeding effort ({rho}= 0.36, p= 0.0181) but less so during subsequent feeding ({rho}= 0.06, p= 0.6811). The median number of expelled sporozoites was of similar magnitude during the first and second feeding event, with a strong correlation between them ({rho}= 0.56, p= 0.0002). We conclude that repeated feeding results in repeated sporozoite inoculation at similar intensity.

microbiology↗

bioRxiv2024

Gametocyte production and transmission fitness of African and Asian Plasmodium falciparum isolates with differential susceptibility to artemisinins

The emergence of Plasmodium falciparum parasites partially resistant to artemisinins (ART-R) poses a significant threat to recent gains in malaria control. ART-R has been associated with PfKelch13 (K13) mutations, which differ in fitness costs. This study investigates the gametocyte production and transmission fitness of African and Asian P. falciparum isolates with different K13 genotypes across multiple mosquito species. We tested three ART-sensitive (ART-S) isolates (NF54, NF135, NF180) and three ART-R isolates (ARN1G, 3815, PAT-023) for sexual conversion and transmission to Anopheles stephensi, An. gambiae and An. coluzzii. ART-R levels were quantified in vitro using the Ring-stage Survival Assay (RSA), and the transmission-reducing effects of dihydroartemisinin (DHA) on mature gametocytes were assessed. Results showed that ART-S parasite lines consistently produced gametocytes and transmitted effectively in all three mosquito species. ART-R isolates showed variability: ARN1G maintained high transmission levels, whereas 3815 showed limited transmission potential despite higher sporozoite loads in An. coluzzii. The African ART-R isolate PAT-023 demonstrated low gametocyte commitment but was transmitted efficiently in both An. gambiae and An. coluzzii. DHA exposure reduced mosquito infectivity for all isolates, regardless of K13 genotype. These findings, based on a limited number of field isolates, suggest that ART-R parasites remain transmissible across different Anopheles species. However, ART-R does not appear to confer a direct transmission advantage. This study highlights the complexity of ART-R dynamics and underscores the need for further research to inform malaria control strategies in regions where ART-R parasites are circulating.

microbiology↗

bioRxiv2024

AlbuMAX supplemented media induces the formation of transmission-competent P. falciparum gametocytes

Asexual blood stage culture of Plasmodium falciparum is routinely performed but reproducibly inducing commitment to and maturation of viable gametocytes remains difficult. Culture media can be supplemented with human serum substitutes to induce commitment but these generally only allow for long-term culture of asexual parasites and not transmission-competent gametocytes due to their different lipid composition. Recent insights demonstrated the important roles lipids play in sexual commitment; elaborating on this we exposed ring stage parasites (20-24 hours hpi) for one day to AlbuMAX supplemented media to trigger induction to gametocytogenesis. We observed a significant increase in gametocytes after AlbuMAX induction compared to serum. We also tested the transmission potential of AlbuMAX inducted gametocytes and found a significant higher oocyst intensity compared to serum. We conclude that AlbuMAX supplemented media induces commitment, allows a more stable and predictable production of transmittable gametocytes than serum alone. HighlightsGametocytes are formed when asexual parasites commit to sexual differentiation. Sexual commitment can be promoted by environmental stressors in media formulations. Short exposure of young asexual parasites to the serum substitute AlbuMAX achieves high proportion of committed gametocytes that are transmission-competent.

microbiology↗

bioRxiv2023

A transmission bottleneck for malaria? Quantification of sporozoite expelling from laboratory and natural P. falciparum infections

It is currently unknown whether all Plasmodium falciparum infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. Sporozoites were quantified by highly sensitive qPCR; intact and ruptured oocysts by fluorescence microscopy following antibody staining of circumsporozoite protein. In laboratory conditions, higher total sporozoite burden in mosquitoes was associated with a shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of P. falciparum infected An. stephensi mosquitoes expelled sporozoites into artificial skin. The medians of expelled and residual salivary gland sporozoites were 136 (IQR: 34-501) and 23,947 (IQR: 9127-78,380), respectively. There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load ({rho}=0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and the number of sporozoites expelled ({rho}=0.35; p=0.0002). In Burkina Faso, An. coluzzii mosquitoes were infected by natural gametocyte carriers. Among mosquitoes that were salivary gland sporozoite positive, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR: 171-2969) and harbored a median of 45,100 residual salivary gland sporozoites (IQR: 20,310-164,900). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load ({rho}=0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled ({rho}=0.7; p<0.0001). Mosquito salivary glands in Burkina Faso harbored 1-3 distinct parasite clones; several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

microbiology↗

bioRxiv2023

Comparative assessment of mosquito size and receptiveness to P. falciparum infection of two rearing procedures for Anopheles mosquitoes

Malaria is transmitted when Anopheline mosquitoes ingest Plasmodium parasites during blood-feeding. Artificial feeding assays allow mosquitoes to take up blood from membrane feeders, and are widely used to study malaria transmission. These assays require large quantities of mosquitoes; insectaries optimize their rearing procedures to generate high yields of permissive, homogeneous mosquito populations. Rearing of Anopheline stephensi mosquitoes was protocolized at the Radboudumc in the 1980s, yet infection outcomes remain heterogeneous. This study explores possible improvements in mosquito rearing to improve homogeneity of the resulting mosquito populations. It compares the current mass-rearing standard with an adapted alternative approach from another institute that optimizes larval density per tray and applies a diet that has previously been reported. Differences between procedures were assessed by measuring mosquito size by proxy of wing-length and imbibed blood meal volume. To assess receptiveness to P. falciparum infection mosquitoes were fed cultured gametocytes. We observed a slight decrease in mosquito size when applying the alternative rearing procedure, but generated equally parasite-receptive P. falciparum mosquitoes compared to the standard procedure. We conclude that both rearing protocols can be used to generate susceptible mosquitoes for conducting malaria research.

systems biology↗

bioRxiv2020

Zonal human hepatocytes are differentially permissive to Plasmodium falciparum malaria parasites

Plasmodium falciparum (Pf) is a major cause of malaria. The mosquito-borne parasite asymptomatically infects hepatocytes in the liver. The resulting schizonts undergo massive replication to generate blood-infective merozoites. Liver lobules are zonated: hepatocytes in different zones perform differential metabolic functions. In search for specific host conditions that affect infectability, we studied the Pf parasite liver stage development in relation to the metabolic heterogeneity of fresh human hepatocytes. We show selective preference of different Pf strains for a minority of zone 3 hepatocytes characterized by the particular presence of glutamine synthetase (hGS). Parasite schizont growth is significantly enhanced by hGS uptake early in development, which showcases an import system at this stage of the parasite life-cycle. In conclusion, Pf development is strongly determined by the differential metabolic status in hepatocyte subtypes. These findings underscore the importance of detailed understanding of hepatocyte host-Pf interactions and may delineate novel pathways for intervention strategies.

microbiology↗

bioRxiv2017

Probabilistic data integration identifies reliable gametocyte-specific proteins and transcripts in malaria parasites

Plasmodium gametocytes are the sexual forms of the malaria parasite essential for transmission to mosquitoes. To better understand how gametocytes differ from asexual blood-stage parasites, we performed a systematic analysis of available omics data for P. falciparum and other Plasmodium species. 18 transcriptomic and proteomic data sets were evaluated for the presence of curated \"gold standards\" of 41 gametocyte-specific versus 46 non-gametocyte genes and integrated using Bayesian probabilities, resulting in gametocyte-specificity scores for all P. falciparum genes.\n\nTo illustrate the utility of the gametocyte score, we explored newly predicted gametocyte-specific genes as potential biomarkers of gametocyte carriage and exposure. We analyzed the humoral immune response in field samples against 30 novel gametocyte-specific antigens and found five antigens to be differentially recognized by gametocyte carriers as compared to malaria-infected individuals without detectable gametocytes. We also validated the gametocyte-specificity of 15 identified gametocyte transcripts on culture material and samples from naturally infected individuals, resulting in eight transcripts that were >1000-fold higher expressed in gametocytes compared to asexual parasites and whose transcript abundance allowed gametocyte detection in naturally infected individuals. Our integrated genome-wide gametocyte-specificity scores provide a comprehensive resource to identify targets and monitor P. falciparum gametocytemia.

microbiology↗