D3266: Crithidia bombi fluorescence loss in the absence of neomycin

The use of fluorescently tagged microparasites is a powerful tool for understanding the location, intensity, and progression of live infection from within the host. To maintain fluorescence, microparasite cells in vitro are cultured in media containing selecting agents, such as antibiotics. However, when observing infection in vivo the host environment is free of this selecting agent, meaning that observing infection progression over time via fluorescence microscopy may not accurately quantify total cell count. 

To understand how fluorescence is lost over time in tagged microparasites, we measured the loss of fluorescence of red fluorescent protein (RFP) in a tagged Crithidia bombi cell line, a common gut parasite of bumble bees. All RFP-tagged C. bombi cells were maintained in replicates of three in media with and without the selecting antibiotic, neomycin. A sample was prepared from each cell flask, then imaged and analyzed to determine the proportion of red fluorescence cells to non-fluorescent cells. In total, each flask was imaged at 10 time points across 22 days using a confocal microscope with both red fluorescence and brightfield channels. 
Our results showed that fluorescence remained stable over the observation period in all flasks, with or without neomycin, suggesting that RFP is a stable marker with no significant selection pressure against its expression. Fluorescence was significantly lower in media without antibiotic, but also remained stable over time further reinforcing that RFP persists even without the selecting agent. This study supports experiments in fluorescence microscopy that are conducted utilizing fluorescently tagged C. bombi cells.