Early mosquito immune patterns correlate with differential Plasmodium falciparum ookinete load

The innate immune response of Anopheles mosquitoes is closely associated with the successful development of Plasmodium parasites and their subsequent transmission to humans. However, the extent to which mosquito immune activation correlates with parasite burden during early sporogonic development, and how additional factors may be linked to variation in this early immune response, remains only partly understood. In this study, we examined the immune responses of individual Anopheles coluzzii mosquitoes fed on Plasmodium falciparum infected blood with identical gametocytemia, yet exhibiting different infection intensities at the ookinete stage. We focused on the critical window of ookinete maturation and midgut traversal (12 to 36 hours post-infection), a key phase for parasite establishment, while minimizing confounding factors such as mosquito age, microbiota composition, blood meal variability, and experimental noise. Ookinete burden was estimated in individual mosquitoes by quantifying four well established stage-specific transcripts (ctrp, warp, soap, and cht1), allowing stratification into "low" and "high" ookinete load groups prior to RNA-seq analysis. Transcriptional profiling revealed clear differences between these two conditions, with mosquitoes carrying lower ookinete loads exhibiting stronger upregulation of immune-related genes compared to those with higher burdens, indicating that the reduced parasite establishment during the very early stages of infection is strictly associated with an effective immune activation. Notably, genes involved in L-arginine homeostasis were significantly modulated, highlighting a pathway not extensively explored in the context of mosquito anti-Plasmodium defense. Overall, our findings show that ookinete burden correlates with distinct mosquito transcriptional states during early infection and emphasize the value of ookinete-specific transcripts quantification for dissecting early vector-parasite interactions.