In-vitro survival and enlargement of Gregarina polymorpha and attempts to generate an invertebrate gut cell line

Gregarines are a large group of ancestral Apicomplexan parasites that exclusively infect invertebrate hosts. Their basal phylogenetic positioning means their study could help to elucidate the evolution of obligate parasitism from free-living photosynthetic ancestors. Yet their lack of clinical significance to humans means little research is dedicated to gregarines and the lack of a culturing system for these organisms dramatically impedes that which is. Due to the inclusion of a life cycle stage in most gregarines which necessitates attachment to host cells for development to progress, a culturing system must incorporate appropriate host cells. The present thesis describes attempts to develop a gregarine host gut cell line, to be used as a culturing platform as well as attempts to sustain three gregarine species. For these cultures a variety of conditions were tested, including temperature, culture medium, antimicrobial concentration, and the type of culturing vessel. Microfluidics were even employed for seven gregarine cultures to simulate the environment of an invertebrate gut more accurately. However, none of the 91 primary gut cell cultures that were created displayed growth or replication though most were attached to the culture vessel base. Further, only Gregarina polymorpha, hosted by Tenebrio molitor larvae, survived, and grew in-vitro. Additionally, during attempts to sequence gregarines from freshwater insect larvae many micrographs were obtained depicting various developmental stages of both Pileophalus sinesis hosted by the caddisfly Brachycentrus subnubilis, and Enterocystis ephemerae hosted by the mayfly Ephemera danica.