The application of ultrastructure expansion microscopy reveals the apical microtubule architecture of Babesia

Tick-transmitted apicomplexan parasites of the genus Babesia infect vertebrate red blood cells and cause babesiosis in humans and animals. Detailed studies of their subcellular organization have been constrained by their small size and intracellular development. In this study, we report the identification of a previously unrecognized apical tubulin rosette (ATRo) in Babesia divergens - a unique rosette-like microtubule structure located at the apical pole of the parasite. The ATRo consists of 6-7 radial spines and represents a distinct architecture that appears morphologically different from the conoid of Toxoplasma and the apical polar rings described in Plasmodium. This discovery was enabled by applying Ultrastructure Expansion Microscopy (U-ExM) to Babesia. U-ExM achieved a 4.3-fold isotropic expansion of the parasite and allowed nanoscale visualization of internal structures using conventional fluorescence labeling and standard confocal microscopy. Using tubulin-specific antibodies, we obtained preliminary insights into the tubulin architecture of the B. divergens apical end. U-ExM provided resolution comparable to transmission electron microscopy (TEM), while offering easier sample preparation, higher throughput and compatibility with proteomic markers, including multiplex immunostaining. These findings establish U-ExM as a powerful new tool for studying Babesia ultrastructure and developmental dynamics and offer a new entry point for exploring invasion mechanisms and potential therapeutic targets.