Two trypanosomatid species,
Lotmaria passim
and
Crithidia mellificae
, have been shown to parasitize honey bees to date.
L. passim
appears to be more prevalent than
C. mellificae
and specifically infects the honey bee hindgut. Although the genomic DNA has been sequenced, the effects of infection on honey bee health and colony are poorly understood. To identify the genes that are important for infecting honey bees and to understand their functions, we applied the CRISPR/Cas9 system to establish a method to manipulate
L. passim
genes. By electroporation of plasmid DNA and subsequent selection by drug, we first established an
L. passim
clone expressing tdTomato or Cas9. We also successfully disrupted the endogenous
miltefosine transporter
and
tyrosine aminotransferase
genes by replacement with drug (hygromycin) resistant gene using the CRISPR/Cas9-induced homology-directed repair pathway. The
L. passim
clone expressing fluorescent marker, as well as the simple method for editing specific genes, could become useful approaches to understand the underlying mechanisms of honey bee-trypanosomatid parasite interactions.