D3121: A comparative analysis of disruptive protocols applied to the protozoan parasite Ophryocystis elektroscirrha

The Neogregarine Protozoan Ophryocystis elektroscirrha is a parasite with a life cycle closely intertwined with that of it host D. plexippus and Danaus gilippus Bernice, the Monarch and Florida Queen butterfly respectively. Previous attempts at genomic studies of O. elektroscirrha proved unsuccessful due to the parasites seemingly unbreakable spore wall. In this project we completed various standard and novel disruption trials assessing and comparing the effectiveness of each protocol at releasing O. elektroscirrha DNA. Individual experiments consisted of systematic thermal cycling, bead beating, sonication and enzymatic trials as well as protocols involving multiple of the above listed disruption methods. While the composition of the O. elektroscirrha spore wall is unknown, inferences from the structures of other Conosidasida species lead to the use of Lyticase, Chitinase, Proteinase K, and ß- Glucuronidase in the enzymatic trails. We produced quantitative measurements of spore disruption as well as photographic evidence of spore lysis. Our study suggests the most effective method for disrupting the spore wall of O. elektroscirrha involves precursory enzymatic treatment followed by bead beating. Such trials provided the clearest photos of spore lysis. The results of this project will allow for a future comparative genomic assay of O. elektroscirrha with newly discovered O. elektroscirrha-like spores found on European Helicoverpa species. Such assay could produce key insights into parasite-host evolution as well as increasing the diversity of genomic data available on Apicomplexan protozoans.