MORN1 has a conserved role in asexual and sexual development across the apicomplexa.

The gene encoding the membrane occupation and recognition nexus protein MORN1 is conserved across the Apicomplexa. In Toxoplasma gondii, MORN1 is associated with the spindle poles, the anterior and posterior rings of the inner membrane complex (IMC). The present study examines the localization of MORN1 during the coccidian development of T. gondii and three Eimeria species (in the definitive host) and erythrocytic schizogony of Plasmodium falciparum. During asexual proliferation, MORN1 is associated with the posterior ring of the IMCs of the multiple daughters forming during T. gondii endopolygeny and schizogony in Eimeria and P. falciparum. Furthermore, the expression of P. falciparum MORN1 protein peaked in late schizogony. These data fit a model with a conserved role for MORN1 during IMC assembly in all variations of asexual development. An important new observation is the reactivity of MORN1 antibody with certain sexual stages in T. gondii and Eimeria species. Here MORN1 is organized as a ring-like structure where the microgametes bud from the microgametocyte while in mature microgametes it is present near the flagellar basal bodies and mitochondrion. These observations suggest a conserved role for MORN1 in both asexual and sexual development across the Apicomplexa.

A toolbox facilitating stable transfection of Eimeria species.

Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria tenella are described here, together with the identification of optimal parameters for the transfection process. A series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that confers resistance to pyrimethamine, were electroporated into sporozoites of the E. tenella Wisconsin strain and stabilised by selective passage through chickens. Very high transfection efficiencies of up to 25% sporozoites in transient transfection and up to 9% oocysts following a single round of in vivo selection were achieved. Crucial factors include the use of very freshly harvested parasites with the AMAXA nucleofection system (program U33 in a cytomix-buffered reaction) and linearised plasmid DNA. The use of a restriction enzyme mediated integration (REMI) protocol boosted overall efficiency and elevated insertion rate per genome. Successful development of methods to generate and isolate stable populations of transfected Eimeria parasites will now stimulate rapid expansion of reverse genetic studies in this important coccidian.