Separating Chromera velia Zoospores From Culture and Estimating Their Average Motility Speed and Lifespan.

Chromera velia is an apicomplexan alga uniquely positioned as the closest photosynthetic relative to apicomplexan parasites (Sporozoa), which include the human pathogens that cause malaria (Plasmodium) and toxoplasmosis (Toxoplasma). Under favorable conditions, C. velia forms motile zoospores that contribute to dispersal and possibly host interaction. However, zoospores coexist with other developmental stages in culture, making their isolation technically challenging. Previous studies characterized the phototactic behavior of zoospores in several taxa, yet this response has not been used to separate motile zoospores from mixed cultures. Other reported methods for zoospore recovery relied instead on physical or chemical principles such as passive filtration, differential centrifugation, or column-based purification, all of which can compromise zoospore motility and viability through mechanical shear or osmotic changes. To address this limitation, we developed a non-invasive, simple, and effective method for rapid zoospore isolation depending entirely on their negative phototaxis response. Using a directional light gradient, the method enables reliable collection of active, motile zoospores without specialized equipment or chemical treatments. Our protocol is straightforward to reproduce, relies on standard laboratory equipment, can be completed in under two hours, and yields a zoospore fraction of sufficient quality for live-imaging, motility assays, and downstream molecular and -omics applications. It may also be adapted to other flagellated protists with light-responsive motile stages. Key features • Requires basic experience with algal culturing and access to a light-controlled environment. • A typical 7-day pre-cultivation period is needed for Chromera velia to initiate and reach peak zoosporogenesis. • Provides a rapid procedure for isolating C. velia zoospores that can be fully completed within 1-2 h, including material preparation. • Enables downstream experiments with clean, motile zoospore populations for physiological, behavioral, molecular, or -omics analyses.