Video acquisition and analysis of trypanosome motility

Tsetse flies were dissected at different infection time points and used for video acquisition. For high-speed analysis the whole alimentary tract including the salivary glands was dissected and spread lengthwise on a glass slide in PBS and covered with a cover slip. Live cell microscopy was performed at room temperature, with an inverted fully automated DMI6000 wide-field microscope (Leica Microsystems, Mannheim, Germany), equipped with a 100x oil and a 63x glycerol objective. Parasites were recorded inside the intact issue or were expelled from the tissue and released in the surrounding PBS. For high-speed recording the sCMOS camera pco.edge (PCO, Kelheim, Germany) was used at frame-rates of 100–250 fps. The total duration of imaging did not exceed 30 min.

For single cell analysis selected sequences were processed with Fiji or Amira. The swimming speed was calculated after measuring the covered distance in the direction of the cell´s movement as described previously (Bargul et al., 2016).

For cellular waveform analysis we choose representative videos of trypanosomes isolated from the fly and swimming persistently forward with uninterrupted tip-to-base beats. Date processing was performed as described previously (Bargul et al., 2016). Cell tracking was performed using the Imaris Software package v8 (Bitplane, Zürich, Switzerland).