Unlocking new understanding of Plasmodium sporozoite biology with expansion microscopy

Transmission of malaria relies on the formation of sporozoites in the mosquito midgut and their subsequent invasion of the salivary gland. Despite their importance, our understanding of the cell biology of sporozoite formation and salivary gland invasion is limited. Here, we apply a technique called Mo squito Tiss ue U ltrastructure Ex pansion M icroscopy (MoTissU-ExM), which physically expands infected mosquito tissues while preserving both host and parasite ultrastructure. Using MoTissU-ExM we are able to observe a range of parasite structures and organelles including features previously seen only by electron microscopy as well as structure not observed before. We leverage MoTissU-ExM to investigate a number of cell biology events during sporozoite formation and salivary gland invasion. In particular we focus on the rhoptries, a secretory organelle important for host cell invasion. We establish a timeline for sporozoite rhoptry biogenesis, show that two rhoptries are used up during salivary gland invasion, and provide the first evidence that rhoptry pairs are specialized for different invasion events. Building on this new knowledge, we characterize the rhoptry protein RON11 and identify it as the first protein involved in sporozoite rhoptry biogenesis. Disruption of RON11 led to the production of sporozoites that specifically fail to invade the salivary gland epithelial cell, thereby blocking transmission of these parasites.