Genetic modification of the bee parasite Crithidia bombi for improved visualization and protein localization

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.