Oocyst wall protein EtOWP6 is critical for sporocyst formation and developmental progression in Eimeria tenella

Transmission of apicomplexan parasites-including Eimeria, Toxoplasma, and Cryptosporidium-relies on the environmentally resilient oocyst wall, a bilayered structure whose integrity is maintained by cysteine-rich oocyst wall proteins (OWPs). Yet the functional roles of these conserved proteins remain poorly defined. Here, we characterize EtOWP6, a conserved OWP in Eimeria tenella, the causative agent of avian coccidiosis. Immunofluorescence reveals that EtOWP6 localizes specifically to wall-forming bodies and oocyst/sporocyst walls during sexual development. CRISPR/Cas9-mediated knockout demonstrates that EtOWP6 is essential for sporocyst formation, with deficient parasites exhibiting malformed sporocysts, developmental arrest in vitro, and an in vivo reduction in oocyst output exceeding 85%. The knockout strain is highly attenuated yet retains partial immunogenicity. Transcriptomic analysis reveals that EtOWP6 deletion induces widespread transcriptional reprogramming, disrupting genes critical for developmental regulation and oocyst wall biogenesis. Our findings establish EtOWP6 as a critical regulator coupling structural integrity with developmental progression in Eimeria, and more broadly, highlight OWPs as conserved targets for transmission-blocking interventions across apicomplexan parasites.