Identification of feeding apparatus components in a heterotrophic marine flagellate

Diplonemids are highly abundant heterotrophic single-celled flagellates that are widespread in the world's ocean. They have a highly complex microtubule-based feeding apparatus (cytostome-cytopharynx complex) located adjacent to the deep flagellar pocket from which two flagella emerge. The apical papilla is a tongue-shaped structure unique to diplonemids that connects the cytopharynx and the flagellar pocket, the latter of which is formed by reinforcing microtubules (MTR) and two flagellar roots called intermediate and dorsal roots. Here we report identification of 17 proteins that localize at the feeding apparatus or flagellar apparatus in Diplonema papillatum. Using ultrastructure expansion microscopy, we show that Mad2 and its interaction partner MBP65 localize at the MTR, intermediate root, and dorsal root. Homologs of proteins that associate with the flagellar apparatus in Trypanosoma brucei (PFR1/2, KMP-11, BILBO1) localize at the feeding apparatus in D. papillatum. We also identify proteins that localize at the apical papilla, MTR, parallel microtubule loop, or cytopharynx. By discovering many components of the feeding apparatus in diplonemids, this work forms the foundation to understand the feeding mechanism in these highly abundant marine planktonic organisms.