Fig. 2.

Fig. 2.

Toxoplasma gondii tachyzoites reside within zebrafish brain cells and neurons. (A) 3D CLEM of tachyzoites in the HBV of transgenic mpeg1:G/U:mCherry larvae harboring macrophages (red) infected with type I Toxoplasma-GFP (green) at 6 hpi. 3D reconstructions of 40 confocal z-slices of a full vibratome section (FM, fluorescence microscopy; top left) and of 354 inverted consecutive 50 nm SBF SEM slices of a segment of it (top right). A middle slice of each of the Toxoplasma visible in the SBF SEM dataset was manually segmented (green; top right) to aid correlation. Regions of interest showing the localization of the high-resolution SBF SEM images (bottom row) are denoted with color boxes. Single (left; green box), replicating (middle; red box) and doublet (right; blue box) tachyzoites in zebrafish host cells were observed. See also Movie 2. Shown are three representative images out of 36 total Toxoplasma in zebrafish brain cells (see Fig. S2); tachyzoites were imaged in their whole volume to accurately determine their stage. Host mitochondrial recruitment to the parasitophorous vacuole is indicated by yellow arrowheads. Scale bars: 10 µm (top row) and 1 µm (bottom row). (B) Representative AiryScan confocal images of 3 dpf Tg(elavl3:GCaMP6 s)^jf4 larvae (neurons marked in green) infected in the HBV with type I Toxoplasma-Tomato (red) at 4 hpi. Shown are maximum projections of 35 z-slices (covering 5.98 µm) out of 85 slices imaged (left, five tachyzoites total imaged). The ventricular surface is highlighted by a white dashed line. Of the three tachyzoites found within green neurons from the left image, shown are magnified maximum-projection images of two tachyzoites covering 8 (i, top right) or 17 (ii, bottom right) z-slices (z=0.17 µm) out of 85 total. Scale bars: 5 µm (left image) and 2 µm (right images). Two of the five tachyzoites imaged are not inside green neurons (left) and close to the ventricular surface where green neurons become sporadic (see Movie 3), which suggests active invasion of progenitors/ependymal cells.