Crithidia bombi Fluorescence Loss in the Absence of Neomycin

Presenter

Neida O. Portillo

Campus

UMass Amherst

Sponsor

Lynn Adler, Department of Biology, UMass Amherst

Schedule

Session 4, 2:30 PM - 3:15 PM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board A20, Campus Center Auditorium, Row 1 (A1-A20) [Poster Location Map]

Abstract

The use of fluorescently-tagged microparasites is a powerful tool for understanding location, intensity and progression of live infection from within the host. In order 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 measure 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 proportion of red fluorescence cells to non-fluorescent cells. In total, each flask was imaged at 10 time-points across a 22-day period using a confocal microscope with both red fluorescence and brightfield channels. 

Our results showed that fluorescence is not significantly lost over the observation period across all flasks with or without Neomycin, suggesting that RFP is a stable marker and furthermore that there may not be a significant selection pressure against expressing RFP. This study serves to reinforce fluorescence microscopy experiments conducted utilizing fluorescently tagged C. bombi cells, because our results suggest that RFP tags will persist even in the absence of a selecting agent.

Keywords

bumblebees, microscopy, microparasite , fluorescence , crithidia

Research Area

Biological Organisms

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