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Abstract

Plasmodium parasite development has been extensively studied in blood stages using different volume electron microscopy (vEM) techniques, describing organelle development and segregation in detail. The mosquito stages of the Plasmodium life cycle stage have in comparison been largely understudied using these techniques. Here I present a comprehensive ultrastructural cell atlas, spanning multiple stages of the Plasmodium mosquito life cycle stage. Beginning with an in-depth characterization of ookinete development. A new staging system was established by clustering morphometric parameters from volume electron microscopy (vEM) data reconstructions, enabling the subdivision of developmental progression into distinct stages based on morphometrics and organellar features. This approach allows a novel classification of ookinete maturation and revealed undescribed subcellular features such as novel nuclear and mitochondrial morphologies or the intricate interactions of the apicoplast and micronemes in ookinetes. Following this, resin-embedded and heavy metal stained samples of infected midguts were imaged with μCT and High throughput tomography (HiTT) to locate oocysts within intact tissues. While μCT offered broad anatomical context, HiTT provided sufficient subcellular resolution to distinguish oocyst stages. Using morphological features visible in HiTT data, six oocyst developmental stages were defined. Quantification of over 450 oocysts confirmed the progression of these stages during infection. These were detailed through correlative HiTT and vEM imaging, including serial blockface SEM (SBF-SEM) of chemically fixed, focused ion beam SEM (FIB-SEM) and electron tomography (ET) of high pressure frozen and freeze subtituted samples. Putting sporozoite development and oocyst maturation into context. Finally, whole-mosquito HiTT imaging demonstrated its power in localizing parasite stages within intact mosquitoes. This enabled the detection of midgut oocysts in situ and gland deformation over time. Correlative HiTT and SBF-SEM imaging confirmed that the X-ray dense structures in the salivary glands are sporozoite clusters, further supporting the usefulness of this correlative imaging for host-pathogen interaction studies. With these ultrastructural cell atlases I propose novel classification stages of the oocyst and ookinete stages, recapitulate sporozoite development with a special focus on nuclear biology and aim to provide a foundation for future functional studies exploring the 3D ultrastructure of mutant parasites.