DOI: 10.5061/dryad.hqbzkh1qt
title: Genetic modification of the bee parasite Crithidia bombi for improved visualization and protein localization

description: 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.
description: # Method for genetic modification of *Crithidia bombi*
 [https://doi.org/10.5061/dryad.hqbzkh1qt](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.