Figure 1.
T. solium larval stage α-tubulin distribution.
A photographic composite of images from cysticercus cryosections reveals the localization of α-tubulin using DMI-A mAb as a primary antibody. The fluorescein coupled to the secondary antibody was distributed along the tegumentary wall (T), the subtegumentary layer (SL) and in dots corresponding to flame cells (FC) that were mainly scattered in the folds of the invaginated scolex (SC). Scale bar = 1 mm.
Figure 2.
α-tubulin and F-actin in flame cells.
α-tubulin (green) and F-actin (red) were revealed in flame cells (FC) of the invaginated scolex (SC). Upper panel: α-tubulin in flames (A–C) and nuclei of the cells stained red with propidium iodide (B and C). In B, under Nomarsky illumination, several FC are seen surrounding protonephridial ducts (*). Middle panel: α-tubulin in flames (D, F) and F-actin in FC and myofibers (MF) (E, F). Button panel: A higher magnification (60×) of a cell (G, H) shows the different distribution of α-tubulin and F-actin: the former is in the flame and the later is surrounding an anterior portion of the flame like a belt clasp (CB). Nuclei were stained with DAPI (blue). As seen under Nomarsky illumination (H, I), the cell is embedded in the interstitial matrix (IM) and several cilia can be distinguished (arrowhead) in its ciliary tuft. Scale Bars A and B = 50 µm, C = 10 µm, D–F = 40 µm and G–I = 2.5 µm.
Figure 3.
F-actin and myosin II in the invaginated scolex.
F-actin (green) is shown in green as done in figure 2. Myosin II was revealed in red. A selected field of the invaginated scolex there is a network of myofibers (MF) that contain F-actin or myosin II. Myosin was also found surrounding several cross-sectioned protonephridial ducts (PD) that, in turn, are surrounded by clusters of bright fluorescent green dots, that correspond to the co-localization of F-actin and myosin II on the belt clasps of flame cells (FC). Some FC appear encrusted in myofiber bundles and in the interstitial matrix (IM). Scale bar = 24 µm.
Figure 4.
Reconstruction and 3D visualization of flame cells.
A) Fluorescent staining of F-actin, α-tubulin and nuclei as indicated in Figure 2F. B) A reconstruction and visualization of the image in A after rendering and reconstructing by AMIRA software a stack of ∼20 images. False colors, similar to those seen by fluorescence, were associated to cytoskeletal proteins and nuclei of flame cells (FC) and, in order to reconstruct the interstitial matrix (IM), a yellow color was selected. Rotation (90°) of the focal plane of FC (C–E), after digital cross sectioning of the image in B, is showing that inside of the clasp the F-actin is forming a cage. 3D visualization of the button panel shows that FC are embedded in tunnels formed in the matrix (see Movie S5). Scale bar = 10 µm.
Figure 5.
Transmission electron microscopy of flame cells.
The complete morphology of three ciliated cells (A, B and C) shows their cell bodies attached to the flames trough a structure of cross-striated rootlets (R). In cell bodies, nuclei (N) are large and have several heterochromatic regions. Cytoplasmic regions have microvesicles (V) empty, filled or discharging through membrane invaginations (*) and some cytoplasmic extensions (CE) appear connected with a glycogen storage cell (G). Interactions between FC and TC (Figures B–D) are due to two external (EL) and internal leptotriches (IL). α-tubulin in cilia of the flame is shown because of the distribution of 20 nm gold conjugated secondary antibody after its interaction with DM1-A mAb (D). Observations were performed using a JEOL 1200EX TEM at 60.0 KeV. Scale bars A and B = 1 µm, C = 2 µm and D = 500 nm.
Figure 6.
Ultrastructure of the protonephridial duct and a cross-striated rootlet of a flame cell.
In A, the lumen of a protonephridial duct (PD) contains small rounded electrodense vesicles presumably coming from cilia (C). In B, at the cross-striated rootlet (R), cilia are bound by a fine regular network of microfibrils (small arrows) that cross the region from one side of the FC to the other. The insert is showing a digital magnification of a selected adjacent region of the correspondent basal bodies. Scale bars A = 500 nm and B = 200 nm.
Figure 7.
Scanning electron microscopy of a flame cell.
The micrograph was taken from a cross-sectioned body of the proglottid of an adult tapeworm. In A, a cell body (CB) is proximal to the empty lumen of a protonephridial duct (PD). In B, the cilia of a flame cell (C) are close to extensions from the wall of the PD (arrow). In both images, flame cells (FC) are surrounded by a spongy interstitial matrix (IM). Scale bars A = 1 µm and B = 2 µm.