Rhoptry biogenesis in Plasmodium sporozoites is uncoupled from mitosis and forms distinct pairs.

Malaria transmission relies on sporozoite formation in the mosquito midgut and subsequent salivary gland (SG) invasion. Despite their importance, the cell biology of these processes remains poorly understood. We apply mosquito tissue ultrastructure expansion microscopy (MoTissU-ExM), which physically expands infected mosquito tissues while preserving host and parasite ultrastructure. MoTissU-ExM reveals parasite structures and organelles, including features previously seen only by electron microscopy and novel structures not observed before. We use MoTissU-ExM to investigate sporozoite formation and SG invasion, focusing on rhoptries-secretory organelles critical for host cell invasion. We establish a timeline for rhoptry biogenesis, show that two rhoptries are consumed during SG invasion, and provide the first evidence that rhoptry pairs are specialized for different invasion events. We further characterize RON11 as the first protein involved in sporozoite rhoptry biogenesis; its disruption produces sporozoites that specifically fail to invade SG epithelial cells, blocking parasite transmission.