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. Highlights We 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