Method for Isolation‬ ‪of Myxozoan proliferative stages‬ ‪from fish at high yield and purity:‬ ‪An essential prerequisite for in vitro,‬ ‪in vivo and genomics-based‬ ‪research developments.

Myxozoans are a diverse group of microscopic cnidarian parasites and some representatives are associated with important diseases in fish, in both marine and freshwater aquaculture systems. Research on myxozoans has been largely hampered by the inability to isolate myxozoan parasites from their host tissues. In this study, we developed and optimized a method to isolate the myxozoan proliferative stages of different size and cellularity from fish blood, using DEAE-cellulose ion exchange chromatography. We optimized several parameters and obtained 99–100% parasite purity, as well as high survival and infectivity. Using polyclonal pan-carp blood cell-specific antibodies, we further developed a rapid cytometric assay for quantification of the proliferative stages, not only in highly concentrated DEAE-C isolates but also in dilute conditions in full blood. Early developmental stages of myxozoans are key to parasite proliferation, establishment, and pathology in their hosts. The isolation of these stages not only opens new possibilities for in vivo and in vitro studies, but also for obtaining purified DNA and protein extracts for downstream analyses. Hence, we provide a long-desired tool that will advance the functional research into the mechanisms of host exploitation and immune stimulation/evasion in this group, which could contribute greatly to the development of therapeutic strategies against myxozoans

Taxa-specific activity loss and mortality patterns in freshwater trematode cercariae under subarctic conditions

Cercarial activity and survival are crucial traits for the transmission of trematodes. Temperature is particularly important, as faster depletion of limited cercarial energy reserves occurs at high temperatures. Seasonal climate conditions in high latitude regions may be challenging to complete trematode life cycle during the 6-month ice-free period, but temperature effects on the activity and survival of freshwater cercariae have not been previously identified. After experimentally simulating natural subarctic conditions during warmer and colder months (13 and 6°C), a statistical approach identifying changes in the tendency of cercarial activity loss and mortality data was used to detect differences in three trematode genera, represented by four taxa (Diplostomum spp., Apatemon spp., small- and large-sized Plagiorchis spp.). A strong temperature-dependent response was identified in both activity loss and mortality in all taxa, with Diplostomum spp. cercariae showing the most gradual changes compared to other taxa. Furthermore, whilst activity loss and mortality dynamics could not be divided into ‘fish- vs invertebrate-infecting cercariae’ groups, the detected taxa-specific responses in relation to life-history traits indicate the swimming behaviour of cercariae and energy allocation among larvae individuals as the main drivers. Cercariae exploit the short transmission window that allows a stable continuance of trematodes’ life cycles in high-latitude freshwater ecosystems.</p