Free, amoeboid movement of organisms within media as well as substrate-dependent cellular crawling processes of cells and organisms require an actin cytoskeleton. This system is also involved in the cytokinetic processes of all eukaryotic cells. Myxozoan parasites are known for the disease they cause in economical important fishes. Usually, their pathology is related to rapid proliferation in the host. However, the sequences of their development are still poorly understood, especially with regard to pre-sporogonic proliferation mechanisms. The present work employs light microscopy (LM), electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) in combination with specific stains (Nile Red, DAPI, Phalloidin), to study the three-dimensional morphology, motility, ultrastructure and cellular composition of Ceratomyxa puntazzi, a myxozoan inhabiting the bile of the sharpsnout seabream. Our results demonstrate the occurrence of two C. puntazzi developmental cycles in the bile, i.e. pre-sporogonic proliferation including frequent budding as well as sporogony, resulting in the formation of durable spore stages and we provide unique details on the ultrastructure and the developmental sequence of bile inhabiting myxozoans. The present study describes, for the first time, the cellular components and mechanisms involved in the motility of myxozoan proliferative stages, and reveals how the same elements are implicated in the processes of budding and cytokinesis in the Myxozoa. We demonstrate that F-actin rich cytoskeletal elements polarize at one end of the parasites and in the filopodia which are rapidly de novo created and re-absorbed, thus facilitating unidirectional parasite motility in the bile. We furthermore discover the myxozoan mechanism of budding as an active, polarization process of cytokinesis, which is independent from a contractile ring and thus differs from the mechanism, generally observed in eurkaryotic cells. We hereby demonstrate that CLSM is a powerful tool for myxozoan research with a great potential for exploitation, and we strongly recommend its future use in combination with in vivo stains.

Motility factors are fundamental for parasite invasion, migration, proliferation and immune evasion and thus can influence parasitic disease pathogenesis and virulence. Salmonid enteronecrosis is caused by a myxozoan (Phylum Cnidarian) parasite, Ceratonova shasta. Three parasite genotypes (0, I, II) occur, with varying degrees of virulence in its host, making it a good model for examining the role of motility in virulence. We compare C. shasta cell motility between genotypes and describe how the cellular protrusions interact with the host. We support these observations with motility gene expression analyses. C. shasta stages can move by single or combined used of filopodia, lamellipodia and blebs, with different behaviors such as static adhesion, crawling or blebbing, some previously unobserved in myxozoans. C. shasta stages showed high flexibility of switching between different morphotypes, suggesting a high capacity to adapt to their microenvironment. Exposure to fibronectin showed that C. shasta stages have extraordinary adhesive affinities to glycoprotein components of the extracellular matrix (ECM). When comparing C. shasta genotypes 0 (low virulence, no mortality) and IIR (high virulence, high mortality) infections in rainbow trout, major differences were observed with regard to their migration to the target organ, gene expression patterns and proliferation rate in the host. IIR is characterized by rapid multiplication and fast amoeboid bleb-based migration to the gut, where adhesion (mediated by integrin-β and talin), ECM disruption and virulent systemic dispersion of the parasite causes massive pathology. Genotype 0 is characterized by low proliferation rates, slow directional and early adhesive migration and localized, non-destructive development in the gut. We conclude that parasite adhesion drives virulence in C. shasta and that effectors, such as integrins, reveal themselves as attractive therapeutic targets in a group of parasites for which no effective treatments are known.