Sustained production and purification of <i>Ellipsomyxa mugilis</i> actinospores in a laboratory mesocosm

Abstract The lack of commercial treatments or vaccines against myxozoan parasites underscores the urgent need for a deeper understanding of the parasite infection in the fish and annelid hosts. Yet, progress in this research area is hindered by the lack of in vitro culture systems and the scarce number of in vivo models available. In addition, it is crucial to develop new protocols for the purification of spores and early developmental stages, as contamination of isolated parasite populations with host material remains a major obstacle for downstream biological applications, including the preparation of next-generation sequencing libraries, proteomics, pathology and immunological studies. Here, we lay the foundation for establishing the life cycle of Ellipsomyxa mugilis in an indoor laboratory mesocosm by co-housing thinlip grey mullet Chelon ramada and polychaetes Hediste diversicolor . A sustained infection of H. diversicolor was achieved, providing enduring access to E. mugilis actinospores. A purification protocol for actinospores was also developed using fluorescence-activated cell sorting and the lectin wheat-germ agglutinin in conjunction with 2 viability dyes, DAPI and propidium iodide, yielding a significantly pure parasite population with approximately 98% viability. This work establishes the basis for the development of a new myxozoan in vivo model and provides an effective, simple and rapid procedure for purifying viable E. mugilis actinospores. Together, these advances establish a framework for future studies on actinospore infectivity in the fish host.