Fig 1.
General view of Siedleckia nematoides trophozoites and gamonts.
A-B. An early trophozoite. C. A young trophozoite. D. Composite micrograph of trophozoites attached to the host intestinal epithelium between microvilli and cilia. Note the pores organised in longitudinal rows, two per each flattened side. E. Attached trophozoite with a smooth surface showing the pores organised in rows. F. Attached gamont lacking the pores. G. Two parasites attached to the brush border of the host intestinal epithelium. H. Composite micrograph showing the sequence of movement of a single detached parasite. I. Detached parasite. A, G-H: LM, bright field; B-F, I: SEM. black asterisk–parasite apical end, h–host tissue, n–nucleus, white/grey/black arrows–row of pores.
Fig 2.
Cortex organisation in Siedleckia nematoides.
A. Apical end of a parasite attached to the host enterocyte. Note the well-developed layer of glycocalyx. B. A detail of parasite apical end focusing on organisation of subpellicular microtubules. C. General view of parasite cross-sectioned in the anterior region. D. General view of a parasite cross-sectioned in the middle region. E. The cross-sectioned pellicle with well-preserved and adjacent cortical cytomembranes. F. Longitudinally-sectioned pellicle with obviously separated cortical cytomembranes. G. Cortex of parasite cross-sectioned in the middle region. H. Protoplasmic fracture face of the plasma membrane with pores. I. Fractured plasma membrane and cortical cytomembranes. A, C, G: RR TEM; B, D-F: TEM; H-I: FE TEM. black arrowhead–plasma membrane, black asterisk–rhoptry, black circle–pore, double/paired black arrowhead–IMC, ei–EF of the internal cytomembrane, ep–EF of the plasma membrane, g–glycocalyx, h–host tissue, mi–mitochondria, mv–mucronal vacuole, n–nucleus, pe–PF of the external cytomembrane, pp–PF of the plasma membrane, white arrowhead–subpellicular microtubule, white arrows–pores, x—micronemes.
Table 1.
The sizes and density of IMP in individual fracture faces of pellicle membranes in Siedleckia nematoides.
Table 2.
Density of IMP (particles/μm2) in different apicomplexans.
Fig 3.
Distribution of the pores on the Siedleckia nematoides surface.
A. Detail of pellicle surface with a well visible row of pores. B. Different longitudinally-sectioned vesicular structures connected to the pellicle and corresponding to the pores observed by SEM. C. An almost superficial section of a parasite revealing the pores and vesicles organised in row. D. Superficially-sectioned cortex showing the layer of subpellicular microtubules and a row of pores of various size. E. Fractured pellicle revealing the row of differently sized pores located on the PF of the internal cytomembrane, but not visible at the plasma membrane. F. A general view of the longitudinally fractured pellicle revealing the external cytomembrane with a lateral row of pores and few randomly distributed pores. The large empty arrowheads with labels show the direction towards anterior (an) and posterior (po) parasite ends. The inset shows the fractured pellicle and pores demarcated by black rectangle in more detail. G. Fractured pellicle showing pores organised in rows; few pores are distributed randomly. H. A fragment of fractured pellicle where several rows of variously sized pores are visible. Inset shows a more detailed view of area demarcated by black rectangle, with alternating small and large pores organised in row. A: SEM; B, D: TEM; C: RR TEM; E-H: FE TEM. black arrowhead–plasma membrane, black arrows–additional row of pores, double/paired black arrowhead–IMC, ee–EF of the external cytomembrane, ei–EF of the internal cytomembrane, pe–PF of the external cytomembrane, pi–PF of the internal cytomembrane, pp–PF of the plasma membrane, t–subpellicular microtubules, white arrows–lateral row of pores, white arrowheads–randomly distributed pores, white circles indicate some of the large pores, white rectangle demarcates the doubled row of pores.
Fig 4.
Organisation of the subpellicular microtubules in Siedleckia nematoides.
A. A superficial section of a cortex revealing the pores and subpellicular microtubules being helically twisted along the longitudinal cell axis. B-C. Higher magnification of the longitudinally-sectioned subpellicular microtubules. Note the rows of filamentous structures running parallel to the adjacent microtubules (grey arrows) and filamentous connections with the microtubules (black arrows). D. Cytoplasmic face of the internal cytomembrane with IMP alignments (white arrows) that correspond to the localisation of subpellicular microtubules. E. The pellicle covering the anterior part of parasite, underlain by one continuous and several intermittent layers of subpellicular microtubules sectioned in cross (left) and tangential (right) plane. F. The view (similar to E) of fractured pellicle underlain with several layers of subpellicular microtubules. G. The cross-sectioned cortex in the middle region of parasite, showing the organisation of subpellicular microtubules with cross-linking protein complexes. H. Fractured subpellicular microtubules with cross-linking protein complexes. I. The detail of pellicle covered by a thick glycocalyx layer. J. The high magnification of cross-sectioned microtubules partially revealing the organisation of tubulin protofilaments. K-L. Various views of fractured pellicle revealing the cross-linking protein complexes. A, E, G, J: TEM; B-C, I: RR TEM; D, F, H, K-L: FE TEM. black arrow–filamentous structures around subpellicular microtubules, black arrowhead–plasma membrane, c–cytoplasm, double/paired black arrowhead–IMC, g–glycocalyx, grey arrow–filamentous structures located between individual microtubules, grey arrowhead–protein complexes localised between the plasma membrane and IMC, iti–inner surface of the true (= not fractured) internal cytomembrane, white arrow–protein complex embedded in the IMC, white arrowhead–subpellicular microtubule. Black circles mark some of the large pores. White ellipse encircles the cross-linking protein complexes anchoring the subpellicular microtubules to the internal cytomembrane.
Table 3.
The treatment of living individuals of Siedleckia nematoides with cytoskeletal drugs.
Fig 5.
Organisation of the subpellicular microtubules in Siedleckia nematoides after treatment with cytoskeletal drugs.
A-B. Treatment with 10 mM colchicine for 2 h: A. Attached gamont. B. Cross-sectioned cortex with subpellicular microtubules. C-E. Treatment with 100 mM colchicine for 1 h: C. Attached gamont. D-E. General view (D) and higher magnification (E) of the cross-sectioned cortex with subpellicular microtubules. F-G. Treatment with 10 μM oryzalin for 8 h: F. Attached gamont. G. Cross-sectioned cortex with subpellicular microtubules. H-I. Treatment with 30 μM oryzalin for 7 h: H. Attached trophozoite and gamont. I. Cross-sectioned cortex with subpellicular microtubules. A, C, F, H: SEM; B, D-E, G, I: TEM. black asterisk–parasite apical end, black arrowhead–plasma membrane, double/paired black arrowhead–IMC, h–host tissue, white arrowhead–subpellicular microtubule. White ellipses demarcate the regions with disrupted microtubules.
Fig 6.
Organisation of the cortex in Siedleckia nematoides after treatment with cytoskeletal drugs.
A-B. Treatment with 10 μM JAS for 8 h: A. Attached gamont. B. Cross-sectioned cortex with subpellicular microtubules. C-D. Treatment with 30 μM JAS for 6 h: C. Attached gamont. D. Cross-sectioned cortex with subpellicular microtubules. E-F. Treatment with 10 μM cytochalasin D for 9 h: E. Attached gamont. F. Cross-sectioned cortex with subpellicular microtubules. G-H. Treatment with 30 μM cytochalasin D for 8 h: G. Attached gamont. H. Cross-sectioned cortex with subpellicular microtubules. A, C, E, G: SEM; B, D, F, H: TEM. black asterisk–parasite apical end, black arrowhead–plasma membrane, double/paired black arrowhead–IMC, h–host tissue, white arrowhead–subpellicular microtubule. White rectangles highlight the reduced spacing between microtubule layers.
Fig 7.
Distribution of myosin (TRITC), α-tubulin (FITC) and F-actin (TRITC) in Siedleckia nematoides before and after application of cytoskeletal drugs.
A–G. Non-treated parasites. A-B. Single optical section revealing the localisation of myosin (A) and α-tubulin (B). The inset in B shows a localisation of α-tubulin in a caudal part of an individual from another optical section. C. Composite view showing the co-localisation of myosin and α-tubulin in a single optical section of parasites shown in A-B. The inset shows a co-localisation of myosin and α-tubulin in a caudal part of an individual from another optical section. D. The localisation of α-tubulin in the superficial region of a gamont. E. Co-localisation of α-tubulin and F-actin in parasites of various developmental stages. F. Composite view revealing the co-localisation of α-tubulin and F-actin in a macrogamont and microgamont in a single optical section. G. More superficial optical section revealing the localisation of α-tubulin in macrogamont shown in F. H-J. Co-localisation of α-tubulin and F-actin in parasites treated with colchicine: H. 10 mM colchicine (2 h). I-J. 100 mM colchicine (1 h). K-M. Co-localisation of α-tubulin and F-actin in parasites treated with oryzalin: K. 10 μM oryzalin (8 h). L. 30 μM oryzalin (7 h). M. Labelling of α-tubulin in a trophozoite treated with 30 μM oryzalin (5 h). The inset shows a co-localisation of α-tubulin and myosin in the trophozoite caudal region. Note the patchy distribution of α-tubulin underlying the pellicle, corresponding to the localisation of subpellicular microtubules. A-C, M: CLSM, IFA/Hoechst, methanol fixation; D: CLSM, IFA, methanol fixation; E-F: CLSM, IFA/phalloidin-TRITC, PFA fixation; G: CLSM, IFA, PFA fixation; H-L: CLSM, IFA/phalloidin-TRITC/Hoechst, PFA fixation. black arrowhead–parasite caudal end, black asterisk–parasite apical end, h–host tissue, white arrowheads–tiny longitudinal lines corresponding to the subpellicular microtubules.
Fig 8.
Phalloidin (TRITC) and antibody (FITC) staining of actin in Siedleckia nematoides before and after application of cytoskeletal drugs.
A-D. Non-treated parasites: A-B. Localisation of F-actin with phalloidin in parasites attached to the host tissue. C. Double labelling with phalloidin and specific anti-actin antibody. D. Double labelling with phalloidin (left) and anti-actin antibody (right), image split into two separate channels. E-F. Double labelling with phalloidin (left) and anti-actin antibody (right) in parasites treated with JAS, images split into two separate channels: E. 10 μM JAS (8 h). F. 30 μM JAS (6 h). G-H. Double labelling with phalloidin (left) and anti-actin antibody (right) in parasites treated with cytochalasin D, images split into two separate channels: G. 10 μM cytochalasin D (9 h). H. 30 μM cytochalasin D (8 h). A-B, D-H left: CLSM, phalloidin-TRITC/Hoechst; C: CLSM, IFA/phalloidin-TRITC/Hoechst; D-H right: CLSM, IFA/Hoechst; A-H: PFA fixation. black arrowhead–parasite caudal end, black asterisk–parasite apical end, h–host tissue.