Fig 1.
Surface topology of forebody tegumentary structures in Paradiplozoon homoion adults.
A) Overall view of an adult. The white circles indicate the area of the mouths and the white rectangles the area of the haptors. SEM. B) Ventral view of the forebody, with a subterminal mouth and two apically located, round projections. SEM. C) Micrograph of an opened mouth revealing two buccal suckers and uniciliated sensory structures. SEM. D) The mouth area covered by numerous uniciliated sensory structures and pits. SEM. E) Detail of the mouth border (the area marked by the white rectangle in D). Note the tegument with numerous pits and sensory structures. SEM. F) Detail of the buccal cavity surface enlarged by numerous foliate and tubular digitations. SEM. asterisks–round projections, black arrowheads–pits, black arrows–tubular digitations, bs–buccal sucker, fb 1– forebody 1, fb 2 –forebody 2, hb 1 –hindbody 1, hb 2 –hindbody 2, mo–mouth, white arrowheads–uniciliated sensory structures, white arrows–foliate digitations.
Fig 2.
Forebody wall musculature of Paradiplozoon homoion adults.
A-B) General views of the body wall musculature arrangement. CLSM, phalloidin-TRITC. C-D) Detail of the body wall musculature. CLSM, phalloidin-TRITC (C) and phalloidin-TRITC/DAPI (D). A-B are composite views created by flattening a series of optical sections, while C-D represent single optical sections. black arrowheads–flame cells, bs–buccal suckers, cm–circular muscles, dm–diagonal muscles, lm–longitudinal muscles, mo–mouth opening, pm–perpendicular muscles.
Fig 3.
The main muscular organs in the forebody of Paradiplozoon homoion adults.
A) General view of the forebody. CLSM, phalloidin-TRITC. B-C) The forebody in median plane optical sectioning. Note the muscles forming thin trabeculae in the forebody apical part and the numerous cells with prominent nuclei located between the trabeculae (C). CLSM, phalloidin-TRITC (B) and phalloidin-TRITC/DAPI (C). D) Detail showing the pharynx muscle wall arrangement and the muscles controlling pharynx movement. CLSM, phalloidin-TRITC/DAPI. E-F) Various optical sections of the pharynx showing the transverse trabeculae and pharyngeal cell nuclei. G-H) Apical view of the pharynx. CLSM, Phalloidin-TRITC (E, H) and phalloidin-TRITC/DAPI (F, G). A-H are composite views created by flattening a series of optical sections. black arrowheads–muscles controlling the pharynx, black arrows–muscles controlling the buccal suckers, bs–buccal suckers, dm–diagonal muscles of the body wall, lm–longitudinal muscles of the body wall, ph–pharynx, pm–muscle fibres fixed perpendicularly to the tegument, um–U-shaped muscle bundle of a sucker, white arrowheads–two sensory structures localised in the area of the round projections, white arrows in E, F–trabeculae, white arrows in H–four round structures.
Fig 4.
Forebody of Paradiplozoon homoion adults, with emphasis on the apical sensory and circular structures.
A) The apical part of the forebody, revealing the distribution of sensory structures and a group of circular structures (encircled). The white rectangle indicates the area of the apical part of the pharynx containing four round structures. CLSM, phalloidin-TRIC. The micrograph is a composite view created by flattening a series of optical sections. B) Single medial plane optical section of the forebody. CLSM, phalloidin-TRIC/DAPI. black arrows–muscles controlling the pharynx, bs–buccal sucker, ph–pharynx with four circular openings, white arrowheads–sensory structures, white arrows–muscles controlling the buccal suckers.
Fig 5.
Forebody of Paradiplozoon homoion adults, with emphasis on the muscular, excretory and nervous systems.
A) Total view of the musculature and the main muscular organs of the forebody. CLSM, phalloidin-TRITC/DAPI. B) Detail of two flame cells located above the buccal suckers and the area with apical circular structures (encircled). CLSM, phalloidin-TRITC (the output image is uncoloured). C) Distribution of flame cells in the area around the pharynx and buccal suckers. CLSM, phalloidin-TRITC. D-E) Forebody nerve cords and excretory system. CLSM, IFA-FITC/Hoechst (D) and IFA-FITC (E). A, D and E are composite views created by flattening a series of optical sections, while B and C represent single median optical sections. bc–buccal sucker, black arrowheads–flame cells, black arrows–longitudinal (dorsal and ventral) nerve cords, ph–pharynx, white arrowheads–uniciliated sensory structures, white arrows–transverse connective cords.
Fig 6.
Mouth border of Paradiplozoon homoion adults.
A) Median plane optical sectioning of the forebody. Note the accumulation of α-tubulin associated with the forebody apical part and buccal suckers. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. B) View of the forebody showing the tubulin-rich apical end. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. C) Detail of a uniciliated sensory structure with a raised circular rim and one long cilium. SEM. D-E) A different optical section of the specimen in B) revealing the tubulin-rich border of the mouth opening and the distribution of uniciliated sensory structures. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst (D) and IFA-FITC/Hoechst (E). F) Arrangement of the muscle fibres around the border of the mouth opening. CLSM, phalloidin-TRITC. G) Detail of uniciliated sensory structures in the forebody apical end. CLSM, IFA-FITC/DAPI. A-B, D-E and G are composite views created by flattening a series of optical sections, while F represents a single optical section. black arrow–raised circular rim, black arrowheads–flame cells, bs–buccal suckers, ph–pharynx, white arrow–cilium, white arrowheads–uniciliated sensory structure.
Fig 7.
Forebody innervation in Paradiplozoon homoion adults.
A) Tegumentary ridges and uniciliated sensory structures. SEM. B) Micrograph revealing the distribution of uniciliated sensory structures. CLSM, IFA-FITC. C-D) Longitudinal and transverse connective nerve cords. CLSM, IFA-FITC. E) Detail of the sensory structure, with visible cilium anchoring. SEM. F-G) Arrangement of uniciliated sensory structures and peripheral nerve fibres within the tegumentary ridges. CLSM, IFA-FITC (F) and IFA-FITC/phalloidin-TRITC (G). H) Superficial distribution of uniciliated structures, a view comparable with B). The inset to the right shows a detail of two sensory structures from H) (encircled), while the left inset shows the circular rim rich in F-actin only; both views are magnified five times. CLSM, IFA-FITC/phalloidin-TRITC. A, C, D, F and G are composite views created by flattening a series of optical sections, while H represents a single median optical section. asterisk–cilium, black arrowhead–circular rim, black arrows—longitudinal nerve cords, mo–mouth opening, white arrowheads–uniciliated sensory structures, white arrows–transverse connective cords.
Fig 8.
Visualisation of the excretory system of Paradiplozoon homoion adults using α-tubulin immunolabeling.
A) Micrograph showing the distribution of flame cells and peripheral nerve fibres in the forebody region. CLSM, IFA-FITC. B) Flame cells (encircled) counterstained with Hoechst to indicate the nuclei of terminal cells. Note the green stained ciliated tufts and rootlets. CLSM, IFA-FITC/Hoechst. C) Detail of the flame cells. The barrel non-ciliated part involves both the terminal and adjacent canal cell. CLSM, phalloidin-TRITC. D) Detail of one flame cell with the ciliated tuft of the terminal cell. CLSM, IFA-FITC. E) Detail of two flame cells. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. A and B are single median optical sections, while C and D are composite views created by flattening a series of optical sections. black arrowheads–flame cells, black arrows–roots of tuft cilia, white arrows–transverse connective cords.
Fig 9.
Hindbody of Paradiplozoon homoion adults.
A) Lateral view of both hindbodies with prominent haptors. SEM. B) Detail of tegumentary papillae and the folds covering the middle part of the hindbody. SEM. C) Detail of the tegument with papillae. The micrograph shows the area marked by a white rectangle in B). SEM. D) The haptor, with eight clamps organised in two rows. SEM. E) Musculature of four clamps with operating muscle bundles. Note the distribution of flame cells. CLSM, phalloidin-TRITC. F) 3D visualisation of the clamp sclerites. CLSM, Gomori staining. G) Clamp sclerites. LM, bright field. H) Pair of marginal hooks localised between the two rows of clamps. CLSM, Gomori staining. D, E, F and H represent composite views created by flattening a series of optical sections. black arrowheads–flame cells, black arrows–tegumentary folds, cl–clamps, double white arrows–marginal hooks, h–haptor, white arrowheads–tegumentary papillae, white arrows–extrinsic muscle bundles.
Fig 10.
Hindbody with haptor in Paradiplozoon homoion adults, with emphasis on musculature.
A) Lateral view of the haptor, with one row of four clamps and three lobed structures (one central and two lateral lobes). SEM. B) Muscular haptor equipped with four pairs of clamps with extrinsic muscle bundles and a central lobe. CLSM, phalloidin-TRITC. C) Central lobe of the haptor, with numerous flame cells. Note the muscle arrangement (longitudinal, circular and diagonal). CLSM, phalloidin-TRITC. D) Detail of the massive muscle bundles controlling the clamp. CLSM, phalloidin-TRITC. E) Detail of the clamp musculature surrounded by flame cells. Note the strong F-actin labelling localised in the barrel part of the flame cells. CLSM, phalloidin-TRITC. F) α-tubulin labelling of flame cell ciliated tufts located near the clamps. CLSM, IFA-FITC/DAPI. B-F represent composite views created by flattening a series of optical sections. black arrows–extrinsic muscle bundles, black arrowheads–flame cells, ce–central lobe, cl–clamps, cm–circular muscles, dm—diagonal muscles, la–lateral lobes, lm–longitudinal muscles, white arrowheads–sensory structures.
Fig 11.
Hindbody with haptor in Paradiplozoon homoion adults, with emphasis on innervation.
A) Detail of the haptor central lobe. Note the distribution of uniciliated sensory structures (F-actin) and peripheral nerve fibre endings (α-tubulin). The micrograph shows the area marked by a white rectangle in C). CLSM, IFA-FITC/phalloidin-TRITC. B) Double F-actin and α-tubulin labelling of the region surrounding the two clamps. The micrograph shows the area marked by a red rectangle in C). The dense red structures represent autofluorescence of the clamp sclerites. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. C) General view of the hindbody labelled for α-tubulin and counterstained with Hoechst. CLSM, IFA-FITC/Hoechst. A-C represent composite views created by flattening a series of optical sections. black arrowheads–flame cells, black arrows–innervation of clamps, cl–clamps, white arrowheads–uniciliated sensory structures, white arrows–peripheral nerve fibres.
Fig 12.
Cellular morphology of Paradiplozoon homoion adults stained with hydrochloric carmine.
A) Micrograph showing the forebody region. The white circles indicate some of the putative unicellular glands. The white rectangle demarks the putative glands in the area of apical circular structures. CLSM, output image not coloured. B) Micrograph showing the haptor. The accumulation of putative gland cells is demarcated by white circle. CLSM, output image not coloured. A-B are composite views created by flattening a series of optical sections. asterisks–paired club-shaped sacs, bs–buccal suckers, ce–central lobe, cl–clamps, g–foregut, ph–pharynx.