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.

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.

Malaria infection is initiated when Plasmodium sporozoites are injected by Anopheles mosquitoes into the human skin. These motile parasites must move through the dermal environment to reach blood vessels, and the...

CONCLUSIONS: It is essential to understand how Plasmodium sporozoites sense and respond to the skin microenvironment for designing targeted interventions. Gaining deeper insight into sporozoite behavior within skin tissue-particularly the underlying signaling pathways and gene expression patterns-combined with advances in diagnostic tools and noninvasive imaging techniques targeting the initial skin-stage infection can facilitate the identification of novel therapeutic targets.

Malaria infection is initiated when Plasmodium sporozoites are injected by Anopheles mosquitoes into the human skin. These motile parasites must move through the dermal environment to reach blood vessels, and the...

The Plasmodium circumsporozoite protein (CSP) is the major surface protein of the sporozoite, the infective stage of the malaria parasite. The central repeat region of CSP is the primary immunological target of the sporozoite stage, yet little is known about its structure or function. Here, we show that sporozoite mutants with truncated or scrambled CSP repeats exhibit impaired motility due to altered adhesion site formation and dynamics. Since CSP forms the environment in which the...

BACKGROUND: Highly effective malaria vaccines are crucial to further reduce the burden of malaria. The radiation-attenuated Plasmodium falciparum sporozoite (PfSPZ) Vaccine protects adults; however, there are insufficient efficacy data in child populations. We aimed to assess the safety and efficacy of the PfSPZ Vaccine in children aged 1-12 years in Gabon.

PfSPZ Vaccine is well tolerated and safe, but it did not prevent P falciparum infection in children in Gabon. Whether presumptive treatment during immunisation or more potent PfSPZ vaccines can establ...

PfSPZ Vaccine is well tolerated and safe, but it did not prevent P falciparum infection in children in Gabon. Whether presumptive treatment during immunisation or more potent PfSPZ vaccines can establ...

Sporozoites of Plasmodium falciparum, the deadliest malaria parasite, are transmitted into the skin by infected mosquitoes and migrate to the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that, eventually, enter the bloodstream and invade erythrocytes, leading to malaria. The parasite journey involves cell traversal, where sporozoites transiently enter and exit host cells beginning in the skin, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish liver-stage infection. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host determinants. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining in HC-04 Cas9+ (clone 2B3) cells. Optimized traversal and invasion assays with HC-04 2B3 cells led to a robust cytometric assay suitable for screening human genes involved in P. falciparum infection. As proof-of-concept, we performed a small screen involving disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens, confirming utility of this platform. While no new host factors were identified for malaria parasites in this initial study, we have developed a tractable system for genome-wide CRISPR screens to uncover novel hepatocyte biology and host determinants of infection by liver-tropic pathogens.

Sporozoites of Plasmodium falciparum, the deadliest malaria parasite, are transmitted into the skin by infected mosquitoes and migrate to the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that, eventually, enter the bloodstream and invade erythrocytes, leading to malaria. The parasite journey involves cell traversal, where sporozoites transiently enter and exit host cells beginning in the skin, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish liver-stage infection. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host determinants. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining in HC-04 Cas9+ (clone 2B3) cells. Optimized traversal and invasion assays with HC-04 2B3 cells led to a robust cytometric assay suitable for screening human genes involved in P. falciparum infection. As proof-of-concept, we performed a small screen involving disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens, confirming utility of this platform. While no new host factors were identified for malaria parasites in this initial study, we have developed a tractable system for genome-wide CRISPR screens to uncover novel hepatocyte biology and host determinants of infection by liver-tropic pathogens.

Understanding the immunology of adverse events is challenging because they are rare and often occur without concurrent serum sampling. This study found tha