Developing gregarine apicomplexans as aquatic symbiosis model systems

The apicomplexan phylum contains a vast group of obligatory parasites that encompass significant pathogens like Plasmodium spp. (the agent responsible for causing malaria) and Cryptosporidium spp. (responsible for the diarrhoeal disease cryptosporidiosis). While these medically relevant apicomplexans receive substantial research attention, the majority of the phylum’s diversity, particularly the gregarines, remains understudied despite regularly appearing in high-throughput sequencing surveys. Gregarines colonise various freshwater, marine, and terrestrial invertebrates. However, there are currently only a handful of transcriptomes and genomes available for gregarine species. Our limited knowledge regarding gregarines is partially due to the lack of available culturing techniques. To date, there are no in vitro methods to culture gregarines in a laboratory environment. Current methods are limited to the culturing of the host organisms (mainly terrestrial invertebrates), which can be restricted due to seasonality (collection of hosts), costs (to maintain complex host life cycles) and labour-intensiveness (regular feeding, cleaning, and maintenance of host culture systems). This project focuses on developing the foundations for an in vitro culturing system for gregarines. The study was carried out using the freshwater amphipod host Gammarus pulex. The hosts were collected using a standard kick sampling method in the Water of Leith river, Scotland (55.896010”N, -3.307785”W). Understanding the gregarine prevalence and diversity within this host species was the initial objective of this project. Gregarine species were identified and described based on a variety of methods, such as light microscopy, scanning electron microscopy, trophozoite size measurements, 18S sequence data, and phylogenetic analysis. Three gregarine symbionts, including a newly described species, were identified to reside in the gut of G. pulex. A protocol was established for culturing gregarine-positive and gregarine-negative amphipods, as well as a protocol to determine if an individual G. pulex was infected with gregarines and if so, by which of the three identified species. Additionally, to further understand the interactions between the gregarines and the host gut, the established protocols were used to provide sample material for transcriptomic, metabolomic, and metabarcoding analyses. The transcriptomes of two previously unsequenced gregarine species identified in this study were sequenced. Potentially important genes involved in metabolic pathways were examined to verify what gregarines require to survive within the host and to better understand the relationship between the gregarine and the host. Corresponding metabolites that may play a role in gregarine survival within the host gut were identified using liquid chromatography-mass spectrometry and nuclear magnetic resonance on the gut contents from gregarine-positive and gregarine-negative hosts. Metabarcoding analysis provided further knowledge on the broader microbial community dynamics between gregarine-positive and gregarine-negative hosts. Experiments to culture Gammarus pulex gut epithelial cells were conducted to support the sporozoite life stage of these symbionts in vitro. Overall, the results presented in this thesis lay the groundwork to establish the first propagating in vitro culturing platform for gregarines.