‘Remarkable and suspicious cells’: Uncovering segmentation in Plasmodium oocysts by volume electron microscopy

Abstract

Malaria has plagued humankind for centuries, and even in modern day, continues to affect millions of people worldwide each year. Plasmodium falciparum parasites are the primary causative agent of human malaria, accounting for over 90% of cases. Over its evolutionary history, the parasite has co-adapted strategies within its human and Anopheles mosquito host to ensure efficient and sustained transmission. One of these strategies involves distinct periods of explosive population expansion following bottlenecks in the parasite life cycle, one of which occurs during oocyst development in the mosquito where thousands of infectious daughter cells are formed. Despite the critical importance of this process for onward transmission, relatively little is known about how the oocyst facilitates this massive cytokinetic event. Thus, new studies utilizing innovative tools to shed light on this essential process are deeply needed. In this dissertation, I describe our efforts to leverage an enhanced focused ion beam-scanning electron microscopy platform to visualize key inter-organelle interactions and biological structures during sporozoite segmentation in oocysts. In Chapter 1, I review the current landscape of knowledge about the non-canonical methods of cell division in Apicomplexan parasites with a focus on the more completely studied Plasmodium asexual blood stages of development. I introduce the capability of using volume electron microcopy for detailed studies of parasite ultrastructure and present limitations of applying this strategy to oocysts with traditional commercial technologies. In Chapter 2, I outline a workflow for utilizing pre-commercial focused ion beam-scanning electron microscopy systems for studying cell division in oocysts, including the significant troubleshooting for adaptation of existing image processing and analysis workflows. In Chapter 3, I apply this workflow to image and subsequently reconstruct in 3D key organelles throughout the entire oocyst volume during mid-segmentation, visualizing and quantifying inter-organelle interactions that may play an organizational role during organelle compartmentalization. In Chapter 4, I modify and apply these strategies to a second oocyst during late segmentation to capture the dramatic morphological and organizational changes that occur during oocyst development and uncover novel biology during sporozoite formation. Finally in Chapter 5, I synthesize how information from this study contributes to a growing understanding of key processes underlying Plasmodium segmentation and present directions for future work interrogating this process in oocysts. Altogether, this dissertation significantly expands our knowledge of daughter sporozoite formation in the oocyst and demonstrates the utility of leveraging advanced imaging techniques to build fundamental understandings of basic parasite biology that is critical for disease transmission.