Genetic modification of the bee parasite Crithidia bombi for improved visualization and protein localization
Data files
Apr 30, 2024 version files 17.36 KB
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GFP_hygromycin_growth_curve.csv
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hygromycin_growth_curve.csv
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neomycin_growth_curve.csv
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README.md
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red_cells.csv
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RFP_neomycin_growth_curve.csv
Abstract
Crithidia bombi is a trypanosomatid parasite that infects several species of bumble bees (Bombus spp.), by adhering to their intestinal tract. Crithidia bombi infection impairs learning and reduces survival of workers and overwintering queens. Although there is extensive research on the ecology of this host-pathogen system, we understand far less about the mechanisms that mediate internal infection dynamics. Crithidia bombi infects hosts by attaching to the hindgut via the flagellum, and one previous study found that a nectar secondary compound removed the flagellum, preventing attachment. However, approaches that allow more detailed observation of parasite attachment and growth would allow us to better understand factors mediating this host-pathogen relationship. We established techniques for genetic manipulation and visualization of cultured C. bombi. Using constructs established for Crithidia fasciculata, we successfully generated C. bombi cells expressing ectopic fluorescent transgenes using two different selectable markers. To our knowledge, this is the first genetic modification of this species. We also introduced constructs that label the mitochondrion and nucleus of the parasite, showing that subcellular targeting signals can function across parasite species to highlight specific organelles. Finally, we visualized fluorescently tagged parasites in vitro in both their swimming and attached forms, and in vivo in bumble bee (Bombus impatiens) hosts. Expanding our cell and molecular toolkit for C. bombi will help us better understand how factors such as host diet, immune system, and physiology mediate outcomes of infection by these common parasites.
README: Method for genetic modification of Crithidia bombi
https://doi.org/10.5061/dryad.hqbzkh1qt
This file describes the archived CSV and rmd file(s) associated with the paper
"Genetic modification of the bee parasite Crithidia bombi for improved visualization and protein localization "
by Blyssalyn V. Bieber, Sarah G. Lockett, Sonja K. Glasser, Faith A. St. Clair, Neida Portillo, Lynn S. Adler, Megan L. Povelones
red_cells.csv
date: Date of image sample
flask_letter: A unique alphabetical identifier for each flask
antibiotic: Treatment of presence/absence of antibiotic neomycin in flasks (yes or no)
num_trial_days: The number of days since the start of the trial
red_cells: Number of red fluorescent cells
all_cells: Number of all cells
ratio_red_all: Ratio of red cells to all cells
hygromycin_growth_curve.csv
replicate: Number of replicate (1,2,3)
time: Duration of time between measuring cell density, in hours
no_hyg: Cell densities in cells per mL in control FP-FB medium lacking hygromycin
hyg_5: Cell densities in cells per mL in FP-FB medium containing 5 micrograms/mL hygromycin
hyg_10: Cell densities in cells per mL in FP-FB medium containing 10 micrograms/mL hygromycin
hyg_20: Cell densities in cells per mL in FP-FB medium containing 20 micrograms/mL hygromycin
hyg_40: Cell densities in cells per mL in FP-FB medium containing 40 micrograms/mL hygromycin
hyg_80: Cell densities in cells per mL in FP-FB medium containing 80 micrograms/mL hygromycin
average: Mean of both time and cell densities across the replicates
neomycin_growth_curve.csv
replicate: Number of replicate (1,2,3)
time: Duration of time between measuring cell density, in hours
no_neo: Cell densities in cells per mL in control FP-FB medium lacking neomycin
neo_5: Cell densities in cells per mL in FP-FB medium containing 5 micrograms/mL neomycin
neo_10: Cell densities in cells per mL in FP-FB medium containing 10 micrograms/mL neomycin
neo_20: Cell densities in cells per mL in FP-FB medium containing 20 micrograms/mL neomycin
neo_40: Cell densities in cells per mL in FP-FB medium containing 40 micrograms/mL neomycin
neo_80: Cell densities in cells per mL in FP-FB medium containing 80 micrograms/mL neomycin
average: Mean of both time and cell densities across the replicates
GFP_hygromycin_growth_curve.csv
replicate: Number of replicate (1,2,3)
time: Duration of time between measuring cell density, in hours
no_hyg: Cell densities in cells per mL in control FP-FB medium lacking hygromycin
hyg_80: Cell densities in cells per mL in FP-FB medium containing 80 micrograms/mL hygromycin
average: Mean of both time and cell densities across the replicates
RFP_neomycin_growth_curve.csv
replicate: Number of replicate (1,2,3)
time: Duration of time between measuring cell density, in hours
no_neo: Cell densities in cells per mL in control FP-FB medium lacking neomycin
neo_80: Cell densities in cells per mL in FP-FB medium containing 80 micrograms/mL neomycin
average: Mean of both time and cell densities across the replicates
The ScholarWorks archive also includes CSV files from which the R scripts read in data to be analyzed.
These CSV files were extracted from original data files in XLS or XLSX format. The CSV files are explained above. The CSV files are included in the archive.
(I) General comments on the R scripts
The R script uses the function here() to identify where to look for the CSV data file, so all R scripts and CSV data files should be put into one folder.
(II) Description of script or markdown files
red_cells.Rmd
This R markdown file tests the effect of antibiotic presence or absence and its interaction with number of days since the beginning of trial on the number of red fluorescent cells. This script requires "red_cells.csv" as an input file.