This thesis steps into many unknowns of the evolutionary biology behind invertebrate hosts and their fungal and microsporidian pathogens and contributes to our understanding of them. However, as is typical to science, we find that our efforts to leave no stone unturned in one aspect of the biology of E. muscae resulted in yet more left to be turned. This is particularly true of the mechanisms involved in behavioural manipulation, where our findings end in us needing to investigate the purpose of a virus apparently ubiquitously associated with E. muscae. Furthermore, the finding of microsporidia being present in numerous hosts, leaves us needing to confirm whether the pathogens are being infectious or simply happen to be in a host species through ecological reasons such as predation. The continuation of studies of these understudied groups of pathogens will only keep delivering new answers and more questions about hostpathogen evolutionary ecology.

The first part of this work introduces a calcein quenching assay using LUVs to evaluate divalent cation transport via the ionophore 4-Br-A23187. This assay proves to be a valuable tool for studying metal transport by ionophores and membrane proteins under well-defined conditions. In the second part, the use of GUVs as a model membrane system is discussed to validate the specific lipid-binding properties of probes, enabling subsequent detection of changes in lipid asymmetry in adherent living cells and analysis of the effects of these changes on cellular function. The third part presents a comprehensive study of the effect of different lipid compositions, including headgroup diversity and fatty acid saturation, and buffer ion concentrations on the quality of GUVs. This study aims to refine the future electroformation protocol for GUVs containing membrane proteins. The fourth part focuses on the reconstitution of membrane proteins into GUVs, a promising strategy with associated challenges. Each membrane protein requires a specific lipid composition and ion concentrations, as demonstrated for the plant P-type ATPase AHA2. GUVs containing AHA2 enable direct observation by optical microscopy, providing insights into membrane protein functionality in single vesicle analysis. The final part of the study concentrates on the expression, purification, and reconstitution of lipid flippases, which play a crucial role in maintaining membrane asymmetry for various cellular processes. The plant flippase SNAP-ALA10/ALIS1 is reconstituted into LUVs, ensuring the flippase remains in an active state, paving the way for its subsequent incorporation and visualization in GUVs. These lipid flippase-containing GUVs provide an excellent model system for a detailed functional analysis of flippases.

In summary, this work emphasizes the importance of model systems, particularly GUVs, in advancing our understanding of membrane dynamics, protein activity, and their intricate interplay in complex cellular environments. This enables further characterization of lipid transporters by combining approaches from different publications shown here.