Protococcidian Eleutheroschizon duboscqi, an Unusual Apicomplexan Interconnecting Gregarines and Cryptosporidia

Abstract

This study focused on the attachment strategy, cell structure and the host-parasite interactions of the protococcidian Eleutheroschizon duboscqi, parasitising the polychaete Scoloplos armiger. The attached trophozoites and gamonts of E. duboscqi were detected at different development stages. The parasite develops epicellularly, covered by a host cell-derived, two-membrane parasitophorous sac forming a caudal tipped appendage. Staining with Evans blue suggests that this tail is protein-rich, supported by the presence of a fibrous substance in this area. Despite the ultrastructural evidence for long filaments in the tail, it stained only weakly for F-actin, while spectrin seemed to accumulate in this area. The attachment apparatus consists of lobes arranged in one (trophozoites) or two (gamonts) circles, crowned by a ring of filamentous fascicles. During trophozoite maturation, the internal space between the parasitophorous sac and parasite turns translucent, the parasite trilaminar pellicle seems to reorganise and is covered by a dense fibrous glycocalyx. The parasite surface is organised in broad folds with grooves in between. Micropores are situated at the bottom of the grooves. A layer of filaments organised in bands, underlying the folds and ending above the attachment fascicles, was detected just beneath the pellicle. Confocal microscopy, along with the application of cytoskeletal drugs (jasplakinolide, cytochalasin D, oryzalin) confirmed the presence of actin and tubulin polymerised forms in both the parasitophorous sac and the parasite, while myosin labelling was restricted to the sac. Despite positive tubulin labelling, no microtubules were detected in mature stages. The attachment strategy of E. duboscqi shares features with that of cryptosporidia and gregarines, i.e. the parasite itself conspicuously resembles an epicellularly located gregarine, while the parasitophorous sac develops in a similar manner to that in cryptosporidia. This study provides a re-evaluation of epicellular development in other apicomplexans and directly compares their niche with that of E. duboscqi.

Fig 1. Light microscopic observations on attached stages of Eleutheroschizon duboscqi.

A. Putative zoite of E. duboscqi invading the host intestinal epithelium. LM, bright field. B-C. Early trophozoites within an already formed PS. Note the caudal prolongation of the sac into a tail. LM, bright field. D. Maturing trophozoite. LM, bright field. E. Mature trophozoite. LM, phase contrast. F. A gamont stage. LM, phase contrast. G. Gamont exhibiting a prolonged tail at the PS. LM, bright field. H. Various stages of trophozoites and gamonts attached to the host intestinal epithelium. LM, differential interference contrast. I. A macrogamont after fixation in PFA. Note the separation of PS from the parasite cortex. LM, bright field. J. Detached macrogamont still enveloped by a PS. LM, bright field. K-N. Macrogamonts (K-L) and microgamonts (M-N) in semi-thin sections. LM, bright field, Toluidine blue. arrow—parasite, arrowhead—tail of the PS, asterisk—parasite attachment site, h—host tissue, n—parasite nucleus/nuclei.

Fig 2. Ultrastructural features of Eleutheroschizon duboscqi early development.

A. Early trophozoite in the process of transformation from an attached zoite, already enveloped by a host-derived PS. TEM. B. Early trophozoite. Note the numerous vesicles in the space between parasite and PS, especially in caudal region. TEM. C-E. Young trophozoite. D shows the space between the parasite caudal region and PS; E shows the host parasite interface at the attachment site. TEM. F-H. Maturing trophozoite. G shows an annular joint point of two host membranes; H focuses on the parasite caudal region and PS. TEM. I. Early trophozoite. SEM. J. Young trophozoite. SEM. K. Mature trophozoite. TEM. L. Detailed view of the attachment site of the trophozoite shown in K, focusing on the developing fascicles of filaments and the annular joint point. TEM. a—parasite amylopectin, asterisk—space between the parasite and PS, black arrow—PS, black arrowhead—parasite plasma membrane, black double/paired arrowheads—parasite cytomembranes, c—parasite cytoplasm, cr—crystalloid body, er—parasite endoplasmic reticulum, fa—attachment fascicles, fi—short attachment filaments, g—glycocalyx, h—host cell, mc—host microcilia, mt—parasite subpellicular microtubules, mv—host microvilli, n—parasite nucleus, r—parasite posterior ring, sf—parasite subpellicular filaments, t—tail of the PS, v—vesicles, white arrow—host cell plasma membrane, white arrowhead—dense band, white double arrowhead—base of the PS (membrane of host cell origin), x—forming attachment fascicles.

Fig 3. Fine structure of Eleutheroschizon duboscqi mature trophozoites.

A. Mature trophozoite transforming into a gamont stage. TEM. B. Detailed view of the annular joint point and a well-developed fascicle of filaments. TEM. C. The view of mitochondria and a micropore (white circle) at the attachment site. The inset shows the micropore in detail. TEM. D. Higher magnification of the caudal region. TEM E. Two partially detached, mature trophozoites. SEM. F. The attachment site of a partially detached, mature trophozoite with well-developed fascicles and short filaments. SEM. G. Diagonal section of the apical part of a mature trophozoite. TEM. H-I. Craters left after detachment of mature trophozoites with well-developed attachment fascicles. Flat holes organised in one circle correspond to the developing lobes. SEM. J. A crater left after a trophozoite of more advanced stage as indicated by the presence of one circle of deep holes corresponding to well-developed lobes and one extra lobe starting the formation of a second circle. SEM. a—parasite amylopectin, black arrow—PS, black arrowhead—parasite plasma membrane, black asterisk—space between the parasite and PS, black double/paired arrowheads—parasite cytomembranes, c—parasite cytoplasm, er—parasite endoplasmic reticulum, fa—attachment fascicles, fh—holes in the host tissue left after the fascicles of the detached parasite, fi—short attachment filaments, g—glycocalyx, h—host cell, l—attachment lobe, lh—holes in the host tissue left after the lobes of the detached parasite, m—parasite mitochondria, mv—host microvilli, n—parasite nucleus, p—parasite, sf—parasite subpellicular filaments, white arrow—host cell plasma membrane, white arrowhead—dense band, white asterisk—empty attachment site, white double arrowhead—base of the PS.

Fig 4. Morphology of Eleutheroschizon duboscqi gamonts.

A. Attached gamont. SEM. B. Macrogamont with a large central nucleus. TEM. C. Microgamont with several nuclei. TEM. D. Macrogamont enclosed by host tissue. TEM, RR. E. The PS tail of the macrogamont shown in D. Note the pores and the mucosubstances present in their surroundings. TEM, RR. F. High magnification of the caudal PS part with the tail showing numerous pores. SEM. G. Detailed view of the tail and gamont caudal part. TEM, RR. H. Upper view of an individual with a ruptured PS. SEM. I. The caudal region of a naked individual. SEM. J. High magnification of the interface between the parasite and PS in the area of the tail. TEM, RR. K. Gamont with two tails at the PS. SEM. a—parasite amylopectin, arrow—PS, asterisk—space between the parasite and the PS, black arrowhead—parasite plasma membrane, black double/paired arrowheads—parasite cytomembranes, c—parasite cytoplasm, db—parasite dense bodies, fa—attachment fascicles, g—glycocalyx, h—host cell, l—attachment lobe, ld—parasite lipid droplets, m—parasite mitochondria, mc—host microcilia, n—parasite nucleus, p—parasite, po—pore, s—mucosubstances, t—tail of the PS, v—vesicles, white arrowhead—base of the PS.

Fig 5. Architecture of attachment site of Eleutheroschizon duboscqi gamonts.

A. Host intestinal tissue with a detached gamont revealing its attachment site at the base of PS. SEM. B. Detail of the gamont attachment site. SEM. C. Detailed view of the fascicles of long filaments alternating with short filaments, organised in ring. SEM. D. A detail of attachment fascicles. SEM. E. Host intestinal tissue with an attached parasite and a crater left after detached ones. SEM. F. A detail of crater left after gamont with well-developed attachment fascicles and two circles of lobes. SEM. G. Host epithelium showing the crater left after the parasite detached. TEM. H. A detail of the PS membrane remains covering the crater. TEM. arrow—PS, fa—attachment fascicle of filaments, fh—holes in the host tissue left after the fascicles of the detached parasite, fi—short attachment filaments, l—attachment lobe, lh—holes in the host tissue left after the lobes of the detached parasite, mv—microvilli and cilia of the host enterocyte, p—parasite, white arrowhead—dense band, white asterisk—empty attachment site, white double arrowhead—base of the PS.

Fig 6. Fine structure of the attachment site of Eleutheroschizon duboscqi gamonts.

A. Macrogamont with a ruptured PS. TEM. B. Oblique section of the attachment site. TEM. C. A detail showing the hook-shaped short filaments anchored into the parasite outer cytomembrane. TEM. D-E. The attachment fascicles in a longitudinal section. The subpellicular layer of filaments is localised just beneath the parasite IMC and ends above the fascicles. TEM; D is stained with RR. F. The annular joint point of two host membranes. TEM. G. A detail of the attachment lobe packed with endoplasmic reticulum and mitochondria. Note the cross-sectioned micropore. TEM. H. Detailed view of vesicles connected with the micropores located in the area of attachment lobes. TEM. I. Longitudinal section of a micropore localised at the parasite attachment site. TEM. J. Detailed view of the attachment fascicles of long filaments alternating with short filaments. TEM. K. The basal part of PS showing an accumulation of fine filaments in the host cell cytoplasm surrounding the PS invaginations with attachment fascicles. TEM, RR. a—parasite amylopectin, asterisk—space between the parasite and PS, black arrow—PS, black arrowhead—parasite plasma membrane, black double/paired arrowheads—parasite cytomembranes, c—parasite cytoplasm, db—parasite dense bodies, er—parasite endoplasmic reticulum, fa—attachment fascicle of filaments, fi—short attachment filaments, g—glycocalyx, h—host cell, hf—filaments in host cell cytoplasm, l—attachment lobe, ld—parasite lipid droplets, m—parasite mitochondria, mv—microvilli and cilia of the host enterocyte, n—parasite nucleus, sf—parasite subpellicular filaments, v—parasite vesicle, white arrow—host cell plasma membrane, white arrowhead—dense band, white double arrowhead—base of the PS. Micropores are indicated by white circles.

Fig 7. Fine structure of a parasitophorous sac and pellicle in Eleutheroschizon duboscqi gamonts.

A. Attached gamonts. SEM. B. Cross-sectioned gamont showing its surface organised in 12 broad folds and shallow grooves corresponding to the regularly arranged interruptions of subpellicular filaments. TEM. C. Longitudinal section showing the organisation of PS, gamont pellicle and the subpellicular layer of filaments that is repeatedly interrupted in areas corresponding to the localisation of micropores. TEM, RR. D. Superficial section of the PS and the gamont pellicle. The channel-like structures located in the space between the PS and parasite correspond to the folding of the PS observed under SEM. TEM, RR. E. Tangential section of the gamont surface underlined with subpellicular layer of filaments. TEM, RR. F. Diagonal section of the gamont surface revealing mitochondria connected with micropores. TEM, RR. G. Cross-section of pellicle showing the subpellicular filaments interrupted in the micropore area. TEM, RR. H. Almost longitudinal section of pellicle with interrupted subpellicular filaments. TEM, RR. I. Pellicle with continuous cytomembranes. TEM. J-K. Re-building of the parasite IMC indicated by the discontinuous cytomembranes and numerous vesicles located between the parasite plasma membrane and the subpellicular layer of the filaments. TEM, RR. a—parasite amylopectin, arrow—PS, asterisk—space between the parasite and PS, black arrowhead—parasite plasma membrane, black double/paired arrowheads—parasite cytomembranes, db—parasite dense bodies, er—parasite endoplasmic reticulum, g—glycocalyx, h—host tissue, ld—parasite lipid droplet, m—parasite mitochondria, p—parasite, sf—parasite subpellicular filaments, v—vesicles, white arrowheads—channel-like structures. Micropores are indicated by white circles, interruptions of subpellicular filaments—by white asterisks.

Fig 8. Fluorescent visualisation of an Eleutheroschizon duboscqi parasitophorous sac.

A. Macrogamont stained with Evans blue. CLSM (lower) and CLSM in a combination with transmission LM (upper two). B-E. Trophozoites (B-D) and a gamont (E) stained with Evans blue. CLSM, output image not coloured. F-H. Localisation of F-actin in trophozoites. One circle of lobes is visible in the attachment site of the trophozoite shown in G. CLSM in a combination with transmission LM (F) and CLSM (G, H), phalloidin-TRITC/DAPI. I. F-actin labelling of a putative young microgamont with two primary nuclei. CLSM, phalloidin-TRITC/DAPI. J. F-actin in a microgamont with numerous nuclei. CLSM, phalloidin-TRITC/DAPI. K-M. F-actin in a putative macrogamont. CLSM in a combination with transmission LM (K) and CLSM (L, M), phalloidin-TRITC/DAPI. N-P. F-actin in a macrogamont equipped with attachment lobes organised in two circles. CLSM, phalloidin-TRITC. The intensity of signal for F-actin shown in F-P was strong for PS and medium for parasites. Q. Labelling of F-actin in an individual treated for 9 hours with 10 μM JAS showing the very strong labelling of PS. Individual optical sections also revealed a slightly increased F-actin labelling of the parasite. CLSM, phalloidin-TRITC/DAPI. R. Treatment with 30 μM JAS for 7 hours resulted in further increase of F-actin labelling in the PS, parasite and host tissue. The individual with several nuclei corresponds to the microgamont stage. CLSM, phalloidin-TRITC/DAPI. S. Visualisation of F-actin in an individual (putative young microgamont with two primary nuclei) treated for 9 hours with 10 μM cytochalasin D. Note the strong labelling of PS in contrast to the parasite and host tissue exhibiting only very weak signal. CLSM, phalloidin-TRITC/DAPI. T. Very weak F-actin labelling in a specimen treated for 7 hours with 30 μM cytochalasin D. CLSM, phalloidin-TRITC/DAPI. A-L, N-O, Q-T are composite views created by flattening a series of optical sections, while M and P represent single median optical sections. All samples were fixed in PFA. arrow—tail of the PS, asterisk—parasite attachment site, black arrowhead—PS, h—host tissue, n—parasite nucleus, white arrowhead—parasite pellicle.

Fig 9. Immunolocalisation of Eleutheroschizon duboscqi cytoskeletal proteins.

A-B. Actin labelling with a medium intensity in a trophozoite (PFA fixation). CLSM, IFA (A) and CLSM in a combination with transmission LM, IFA/DAPI (B). B represents a single median optical section. C. Actin labelling in a macrogamont treated with 30 μM JAS for 7 hours (PFA fixation). Note the increased accumulation of parasite actin (FITC) organised in longitudinal bands exhibiting strong fluorescence and strong F-actin (TRITC) labelling with a diffuse character. CLSM, IFA/phalloidin-TRITC. D. A gamont exhibiting a more diffuse actin (FITC) labelling of medium intensity after treatment with 10 μM cytochalasin D for 9 hours (PFA fixation). The F-actin (TRITC) labelling of the parasite did not change significantly. CLSM, IFA/phalloidin-TRITC. E. Very strong myosin (TRITC) labelling restricted to the PS and host tissue (PFA fixation). CLSM, IFA/DAPI. F. Strong spectrin (FITC) labelling of the PS in a macrogamont (PFA fixation). CLSM, IFA/DAPI. Single median optical section. G. Labelling of α-tubulin (FITC) of strong intensity in a young microgamont (PFA fixation). CLSM, IFA/DAPI. H-I. A trophozoite (fixed in ice-cold methanol) exhibiting a labelling of medium intensity for α-tubulin (FITC) and very strong intensity for myosin (TRITC). CLSM, IFA/DAPI. J. Labelling of α-tubulin (FITC) and myosin (TRITC) in an early trophozoite treated for 7 hours with 10 μM oryzalin (fixed in ice-cold methanol). The fluorescence signals for both antibodies did not change significantly. CLSM, IFA. K-L. Localisation of α-tubulin (FITC) and myosin (TRITC) in an individual (probably a young microgamont) treated with 30 μM oryzalin for 3 hours (fixed in ice-cold methanol). The fluorescence signal for tubulin became very weak, while it remained very strong for myosin. CLSM, IFA/DAPI. M. Co-localisation of α-tubulin (FITC) and F-actin (TRITC) in a macrogamont treated for 7 hours with 10 μM oryzalin (PFA fixation). CLSM, IFA/phalloidin-TRITC. N-O. Labelling of α-tubulin (FITC) and F-actin (TRITC) in a maturing trophozoite treated for 3 hours with 30 μM oryzalin (PFA fixation). CLSM, IFA/phalloidin-TRITC/DAPI. In both the preparations (M-O), there was almost no fluorescence signal for α-tubulin, while the F-actin labelled with a strong intensity. arrow—tail of the PS, asterisk—parasite attachment site, black arrowhead—PS, h—host tissue, n—parasite nucleus, white arrowhead—parasite pellicle.

Fig 10. Schematic diagram of host-parasite interactions in Eleutheroschizon duboscqi, eugregarines, cryptosporidia, and epicellular eimeriids.

The diagrams of E. duboscqi, gregarines and cryptosporidia are based on our personal observations enriched by published data. The diagram of eimeriids represents our interpretation and summary of published micrographs, where only maturing or mature stages were clearly shown [–,,– 44,64,65]. In this scheme, we refer to the host-derived envelope (described as a parasitophorous vacuole throughout literature) of eimeriids in epicellular location as a parasitophorous sac (PS) due to its organisation similar to that in cryptosporidia and E. duboscqi. Three colours are used to distinguish between the parasite (in purple), the host cell including its parts modified due to parasitisation (in pink) and the contact zone between the host and the parasite (in yellow) where the interrelationships of the two organisms become more intimate. In the case of host-parasite cellular interactions in E. duboscqi and epicellular eimeriids, the internal space between the parasite and PS remained colourless, even though we do not exclude the possibility that this region may serve as a transitional zone for intensive interactions between the host and its parasite. A-D. Eleutheroschizon duboscqi. A. Attached zoite transforming into a trophozoite stage, already completely enveloped by a PS. B. Maturing trophozoite with a forming ring of fascicles at the attachment site. The tail forms at the caudal part of the PS. C. Mature trophozoite with a prominent tail. Note the presence of attachment fascicles and lobes. D. Detailed view of the annular joint point (the cut-out is marked by a red square in C). E-H. Eugregarines. E. Sporozoite immediately after attachment to the host epithelial cell. F. Transformation of the sporozoite into a trophozoite stage. G. Early trophozoite with a well-developed epimerite. H. Detailed view of the membrane fusion site (the cut-out is marked by red square in G). The two cytomembranes end at the point of membrane fusion, where the osmiophilic ring is formed. I-L. Cryptosporidia. I. Attached zoite transforming into a trophozoite stage, partially enveloped by an incomplete PS. J. Young trophozoite almost completely enveloped by a PS. Note the tunnel connection between the interior of the anterior vacuole and the host cell cytoplasm that developed as the result of the Y-shaped membrane junction. K. Mature stage with a prominent filamentous projection at the base of the PS and with a fully developed feeder organelle, the lamellae of which formed from the anterior vacuole membrane. L. Detailed view of the Y-shaped membrane junction (the cut-out is marked by a red square in K). M-P. Epicellular eimeriids. M. Invading zoite. N. Trophozoite/meront stage enveloped by a PS with a single attachment area (monopodial form). O. Extension of the gamont stage above the microvillous region leading to an establishment of a new contact with the host cell apart from the primary attachment zone by penetration of the PS membrane to the base of fused microvilli (spider-like form). P. Detailed view of the attachment area (the cut-out is marked by a red square in O). av—anterior vacuole, b—epimeritic bud, cm—parasite cytomembranes, cv—epimeritic cortical vesicle, db—dense band (in cryptosporidia usually consisting of several layers), f—membrane fusion site, dl—dense line separating the feeder organelle from the filamentous projection of the PS, fa—attachment fascicle of filaments, fo—feeder organelle with membranous lamellae, fom—membrane limiting the lamellae of feeder organelle, fp—filamentous projection of the PS, fs—flask-shaped structure, hc—host cell, hm—host cell plasma membrane, if—incomplete fusion of PS, int—interface between the host cell and eugregarine epimerite, consisting of host cell plasma membrane, epimerite plasma membrane and a dense layer in between, ipm—inner membrane of the PS, is—internal space between the parasite and PS, j—annular joint point (Y-shaped membrane junction in cryptosporidia), lo—attachment lobe, ms—membrane-like structure limiting the cortical vesicle from the epimerite cytoplasm, opm—outer membrane of the PS, p—pore on the PS, pm—parasite plasma membrane, ps—parasitophorous sac, r—rhoptries, t—tail of the PS, tu—tunnel connection.