In vivo imaging of host-pathogen interactions: Using transparent mosquitoes to study Plasmodium development in real-time

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Malaria is an important vector-borne disease that affects annually millions of people around the globe. The etiological agents of malaria are unicellular parasites of the genus Plasmodium that evolved special adaptions to replicate within and be transmitted by mosquitoes. Both are critical factors for the parasite because the adaption to its vector is the basis for efficient spread to new hosts to complete its life cycle. While the development of Plasmodium in its vertebrate host can be studied in vitro and in vivo by using human or rodent malaria parasites we still lack profound data especially about its migration within the mosquito. This lack of knowledge can be explained by the difficulty to image through the opaque and pigmented exoskeleton of the mosquito but also by the absence of cell culture systems to study the development of insect stages in vitro. Hence, our knowledge about the development of Plasmodium in its vector relies mostly on ex vivo studies and confocal and electron microscopy on fixed samples. Recent progress in the development of the CRISPR/Cas technology now enables fast and efficient genomic engineering of Anopheles mosquitoes that will also improve the study of Plasmodium in its vector. Utilizing this genetic approach, we propose to generate colourless Anopheles mosquito strains that can be used as tool to study Plasmodium in vivo. Initially we will focus on genes known to be important for pigmentation in Drosophila. After depletion of these genes and subsequent evaluation of their phenotypes in Anopheles, we will select a mosquito strain that combines low pigmentation with high viability. Subsequently this strain will be infected with a highly fluorescent Plasmodium parasite line and single steps of its development will be imaged in vivo using state of the art microscopy techniques. By combining engineered mosquitoes with high-end microscopy we propose to image migrations of the malaria parasite within the mosquito in hitherto unknown resolution of space and time.