Parasitol Res (1992) 78:368-375
Parasitology Research
9 Springer-Verlag1992
Ultrastructure of the developing protonephridial system of the cercaria of Philophthalmus sp. (Trematoda, Digenea)* K. Rohde and N.A. Watson Department of Zoology, University of New England, Armidale, NSW 2351, Australia Accepted February 12, 1992
Abstract. The fully developed flame bulb of Philopthalmus exhibits the structure characteristic of Trematoda and Monogenea: external and internal ribs :forming a weir, external and internal leptotriches, and two longitudinal cytoplasmic cords connected by a septate junction. The proximal canal has a septate junction and surface lamellae. In developing cercariae, perikarya of terminal and proximal canal cells are close together, and sheetlike outgrowths of the terminal cell are externally surrounded by cytoplasm of the proximal canal cell containing a septate junction. Internal outgrowths and external cytoplasm are connected by many "membranes", i.e. desmosome-like structures. Internal sheets break up into internal ribs, and the external cytoplasm breaks up into external ribs, external and internal ribs connected by the filtration '~membrane". The developing distal excretory duct possesses a septate junction and many branching and looping lamellae. A comparison of PhilopthaImus with the cestode Austramphilina elongata, the only other platyhelminth species in which the development of the protonephridia has been studied at the ultrastructural level, revealed that the two species differ in the presence and absence, respectively, of a septate junction in the flame bulb at an early stage of development.
The ultrastructure of the protonephridia of free-living and parasitic Platyhelminthes has received much attention, mainly with the aim of contributing to a phylogenetic system of the phylum (reviews by Rohde 1990, 1991 ; references in Rohde et al. 1992). Among the major groups of parasitic Platyhelminthes (Neodermata sensu Ehlers 1985), two types of protonephridia can be distinguished, one characterized by cytoplasmic cords along the weir of the flame bulb connected by a septate junction and by a septate junction in the proximal capillaries, * Financially supported by the Australian Research Council and the University of New England Offprint requests to: K. Rohde
laries, and another without such cords or junction. The first type is found in the Trematoda Digenea, Trematoda Aspidogastrea and Monogenea and the second, in the cestodes, including the Amphilinidea and Gyrocotylidea, as well as in Udonella (Rohde et al. 1992). To date, the development of the protonephridia of only one species has been examined with the electron microscope: that of the amphilinid Austramphilina elongata (see Rohde and Watson 1988). In this paper, we examine the developing protonephridia of the cercaria of Philophthalmus sp. Comparison of the trematode (weir with cytoplasmic cords) with the cestode (weir without cytoplasmic cords) will show whether there are fundamental differences in the development of the protonephridia in the two groups. Materials and methods Rediae containing developing and fully developed cercariae of Philophthalmus sp. were dissected out of Pyrazus obeninus snails collected in Deception Bay near Brisbane in March 1987 and then fixed for 1-2 h in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4~ C. Specimens were washed in the same buffer at 4~ C, postfixed in 1% OsO~ in 0.1 M phosphate buffer (pH 7.2) at room temperature for 30 min, and dehydrated in an alcohol series. They were embedded in Spurr's resin and polymerized overnight at 60~ C. Sections measuring 60-70 nm in thickness were stained with alcoholic uranyl acetate and lead citrate and examined under a Jeol 1200 EX electron microscope operating at 60 kV. Results The fully (or almost fully) developed flame bulb and proximal capillary exhibit the structure characteristic of Trematoda and Monogenea (Figs. 1 5, 23): a terminal cell with a nucleus close to the weir and a proximal canal cell possessing internal and external ribs (rods), respectively, that interdigitate to form the filtration apparatus or weir. Ribs are connected by a filtration " m e m b r a n e " . External processes or leptotriches arise from the external ribs, and internal leptotriches arise
369
Figs. 1-5. Fully or almost fully developed flame bulb. Figs. 1, 3. Longitudinal sections through the flame bulb and proximal capillary (p). Note the perikaryon of the terminal cell (t), the rootlets of cilia (r), and the internal leptotriches (i/). Fig. 2. Longitudinal section through the base of the weir. Note the interdigitating external (er) and internal ribs (it), the former arising from the proximal canal cell and the latter, from the terminal cell (t). Fig. 4. Cross
section through the weir. Note the internal and external ribs connected by a "membrane", the cytoplasmic cords connected by a septate junction (s), and the internal and external (e/) leptotriches. c, cilia. Fig. 5. Cross section through the basal bodies (b) and rootlets (r) of cilia forming the flame. Note the hollow rootlets. Scale bars = 1 gm
370
Figs. 6-9. Sections through the flame bulb and excretory ducts of a cercarial embryo. Figs. 6-8. Developing flame bulb. Note the terminal cell (t), the proximal canal cell (p), the septate junction (s) in the wall of a capillary formed by the canal cell and between the terminal and proximal canal cells, the nucleus (n) of the canal
cell, microtubules (rot), mitochondria (m), and the external and internal (i/) leptotriches. Fig. 9. Excretory duct (ed) near the excretory opening just below the tegument (te) and microtubules (mt). Scale bars = 1 gm
371
Figs. 10-15. Semi-serial cross sections through a developing flame bulb. The cross section shown in Fig. 10 is that most distally located. Note the septate junction (s) between the cytoplasmic cords of the proximal canal cell (p), the sheet-like process of the terminal cell (t) extending into the cylinder formed by the proximal canal cell and connected to it by desmosome-like connections ("membranes", me). The sheet-like processes of the terminal cell break
up into the internal ribs (ir), and the cytoplasm of the outer wall of the cylinder formed by the proximal canal cell breaks up between the attachment zones of the "membranes" (large arrowheads') opposite the internal ribs. On the right of the section, internal and external ribs are arranged in a single row. n, nucleus. Scale bars = 1 pm
372
Figs. 16--22. Sections through a developing flame bulb and excretory duct. Figs. 16-18. Semi-serial oblique sections through the developing flame bulb. Note the terminal (t) and proximal canal cells (p) attached to each other by numerous desmosome-like connections. Also note the basal body (b) in the cytoplasm of the terminal cell, some distance from the base of the flame. Fig. 19. Longitudinal section through an excretory pore (ep) and duct at-
tached by septate junctions (s) to surrounding tissue. Fig. 20. Cross section through the excretory duct. Note the septate junction (s) and numerous branched and looping lamellae extending into the lumen of the duct. Figs. 21-22. Excretory duct (ed) nucleus (n) excretory pore (ep). Note the septate junction around the terminal part of the duct and the lamellae in the duct. Scale bars = 1 pm
373
o
O
Fig. 23A-C. Diagrams of a fully developed flame bulb. A Longitudinal section. B Cross section through the proximal capillary. C Cross section through the weir (filtration apparatus), c, cilium; cc, cytoplasmic cord; el, external leptotrich; er, external rib; il, internal leptotrich; internal rib; l, lamella; me, "membrane" ; n, nucleus; p, proximal canal cell; s, septate junction; t, terminal cell; v, vesicle. Scale bar = 1 lain
from the internal ribs and basal cytoplasm of the terminal cell near the basal bodies of the cilia forming the flame. The basal parts of the cilia are separated by narrow spaces, but they remain in tight contact along most of their length, forming a functional whole. The central pairs of microtubules are identically oriented in all cilia (Fig. 4). The cilia have cross-striated rootlets that are hollow along part of their length (Fig. 5). Cytoplasmic cords connected by a septate junction extend along the weir (Fig. 4). The internal surface of the proximal capillary is enlarged by numerous lamella-like projections (Fig. 1).
Whereas the nucleus of the proximal canal cell in fully developed protonephridia is located at a great distance from the terminal cell, it is very close to it in developing protonephridia (Fig. 8), indicating that the proximal canal cell undergoes considerable stretching during development. Furthermore, whereas the perikaryon of the terminal cell, including most of its cytoplasm, is located basal to the flame (seen in longitudinal sections of at least six terminal cells (Figs. 1, 3) in fully developed protonephridia, the terminal cell body in the developing flame bulb extends around much of the flame (observed in ten cells; Figs. 7, 8, 24). During development, processes of the proximal canal cell maintain contact with the terminal cell and probably with each other via a number of septate junctions (Figs. 6, 7), and microtubules extend in a predominantly longitudinal direction in both the proximal canal and the terminal cells (Fig. 6). Sheet-like outgrowths of the terminal cell are externally surrounded by cytoplasm of the proximal canal cell (Figs. 6, 7, 10M 8), which contains a septate junction and is connected to the outgrowths of the terminal cell by numerous desmosome-like structures with thick, electron-dense ends in the cytoplasm of both the proximal canal cell and the terminal cell (Figs. 7, 10-18). Observations of several developing terminal cells showed that during development, the cytoplasmic sheets of the terminal cell break up into longitudinal ribs. The external cytoplasm between the ends of two desmosomelike structures from one internal rib becomes less dense and finally disintegrates, leaving the external ribs connected to the internal ones by a " m e m b r a n e " of apparently extracellular matrix (Figs. 10-15, 24A). Irregular outgrowths of the internal ribs and cytoplasmic sheets represent the internal leptotriches (Figs. 7, 14). Internal and external ribs are sometimes arranged in a single row (Figs. 11M 5, 24A), although distinctly alternating external and internal ribs are evident in fully developed flame bulbs (Figs. 4, 23). Occasionally, basal bodies are seen in the cytoplasm of the terminal cell at some distance from the base of the flame (Fig. 18). Basal bodies and ciliary rootlets of lateral (non-terminal) flames are present in the (proximal?) canal (Fig. 6). Lamellae were observed in the developing proximal canal (Fig. 6). We did not examine whether more than one distal canal cell is present. However, the perikaryon of one distal canal cell is located at some distance from the excretory pore (Fig. 21). The distal excretory duct in the developing cercaria contains numerous branching and looping lamellae, leaving a reticular lumen (Figs. 9, 1922), and a septate junction or junctions (Figs. 19-22). The terminal part of the duct is connected to the surrounding tegument by a septate junction (Figs. 19, 22), and several microtubules extend around it in the tegument near the surface (Fig. 9).
Discussion
The fully developed flame bulb of P h i l o p h t h a l m u s corresponds to that of eight genera of Digenea, three genera of Aspidogastrea and nine genera of the Monogenea
374 A
mt
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9
O
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o
o
Qo t
Polyopisthocotylea and Monopisthocotylea examined (Rohde et al. 1992) in the presence of two cytoplasmic cords continuous with the proximal canal cell and connected by a septate junction. All genera of the taxa examined in this regard also have a septate junction in the proximal capillary and internal surface lamellae in small capillaries. Only one monopisthocotylean monogenean, Anoplodiscus cirrusspiralis, lacks the cytoplasmic cords and a complete septate junction (both in the flame bulb and the proximal capillary). However, the presence of junctions in larger capillaries and of a rudimentary junction in the proximal capillaries indicate that a complete junction has been secondarily lost. Thus, the features mentioned can be considered to be primary characteristics of Trematoda/Monogenea. On the other hand, Udonella, the two genera of Amphilinidea, one genus of Gyrocotylidea, and seven genera of the Eucestoda
P
Fig. 24A, B. Diagrams of a developing flame bulb. A Cross section through the developing weir. B Longitudinal section through the developing flame bulb. c, cilium; cc, cytoplasmiccord; er, external rib; it, internal rib; l, lamella; rot, microtubule;p, proximal canal cell; n, nucleus; s, septate junction; t, terminal cell. Scale bars = 1 gm
examined lack cytoplasmic cords and a junction in the flame bulb and their capillaries are without junctions; moreover, all of these parasites except Udonella which has smooth-walled capillaries, possess short microvilli instead of lamellae (Rohde et al. 1992). In the only ultrastructural study of the development of the protonephridial system of a platyhelminth, that of the amphilinid AustramphiIina eIongata, Rohde and Watson (1988) have shown that three branches of a com. pletely closed cytoplasmic cylinder (proximal canal cell) enclose the flames of three terminal cells. Processes of the terminal cells grow into the space between the cilia and cylinder, and external ribs are formed at the points of contact between inner outgrowths and cylinders, being connected to the internal ribs by "membranes". In Philophthalmus, the formation of ribs proceeds in a similar fashion, although the inner outgrowths are dis-
375
tinctly sheet-like and (at least usually) do not occur as thin ribs as in Austramphilina. Most importantly, a seprate junction was present at the earliest stages of development observed in Philophthalmus, whereas it was never seen in the cestode. Its occurrence at a very early stage of development in the latter parasite cannot be excluded, but if it occurs at all, it must be of very short duration. Also, the lamellae in the capillaries characteristic of Trematoda/Monogenea and the short microvilli characteristic of the cestodes occur at quite early stages of development in the two species examined. We did not examine whether more than one flame is enclosed by a single proximal canal cell, which is nevertheless likely in view of the observation that flame cells in cercariae are arranged in bundles of at least two. A septate junction located between the terminal part of the excretory duct near the excretory pore and the tegument, such as that found by us in Philophthalmus, has also been demonstrated to occur in the cercaria of Schistosoma mansoni by Ebrahimzadeh and Kraft (1971). Lamellae in the caudal excretory canal are also present in the cercaria of Cryptocotyle lingua, although the interconnections between them cannot be seen in the published figure (Rees 1977).
Acknowledgements. We wish to thank Mr. P. Garlick for making facilities at the Electron Microscope Unit, University of New England, available to us, Mr. R. Porter for developing and contactprinting the negatives, Mr. Z. Enoch for printing the electron mi-
crographs, and Ms. B. Rochester for typing the manuscript.
References Ebrahimzadeh A, Kraft M (1971) Ultrastrukturelle Untersuchungen zur Anatomie der Cercarien yon Schistosoma mansoni: II. Das Exkretionssystem. Z Parasitenkd 36:265-290 Ehlers U (1985) Das phylogenetische System der Platyhelminthes. G. Fischer, Stuttgart New York Rees GF (1977) The development of the tail and the excretory system in the cercaria of Cryptocotyle lingua (Creplin) [Digenea: Heterophyidae] from Littorina littorea (L.). Proc R Soc Lond [Biol] 195:425~452 Rohde K (1990) Phylogeny of Platyhelminthes, with special reference to parasitic groups. Int J Parasitol 20:979-1007 Rohde K (1991). The evolution of protonephridia of the Platyhelminthes. Hydrobiologia 227 : 3/5 321 Rohde K, Watson NA (1988) Development of the protonephridia of Austramphilina elongata. Parasitol Res 74: 255-261 Rohde K, Watson NA, Roubal FR (/992) Ultrastructure of the protonephridial system of Anoplodiscus cirrusspiralis (Monogenea, Monopisthocotylea). Int J Parasitol (in press)
Parasitology Research
9 Springer-Verlag1992
Ultrastructure of the developing protonephridial system of the cercaria of Philophthalmus sp. (Trematoda, Digenea)* K. Rohde and N.A. Watson Department of Zoology, University of New England, Armidale, NSW 2351, Australia Accepted February 12, 1992
Abstract. The fully developed flame bulb of Philopthalmus exhibits the structure characteristic of Trematoda and Monogenea: external and internal ribs :forming a weir, external and internal leptotriches, and two longitudinal cytoplasmic cords connected by a septate junction. The proximal canal has a septate junction and surface lamellae. In developing cercariae, perikarya of terminal and proximal canal cells are close together, and sheetlike outgrowths of the terminal cell are externally surrounded by cytoplasm of the proximal canal cell containing a septate junction. Internal outgrowths and external cytoplasm are connected by many "membranes", i.e. desmosome-like structures. Internal sheets break up into internal ribs, and the external cytoplasm breaks up into external ribs, external and internal ribs connected by the filtration '~membrane". The developing distal excretory duct possesses a septate junction and many branching and looping lamellae. A comparison of PhilopthaImus with the cestode Austramphilina elongata, the only other platyhelminth species in which the development of the protonephridia has been studied at the ultrastructural level, revealed that the two species differ in the presence and absence, respectively, of a septate junction in the flame bulb at an early stage of development.
The ultrastructure of the protonephridia of free-living and parasitic Platyhelminthes has received much attention, mainly with the aim of contributing to a phylogenetic system of the phylum (reviews by Rohde 1990, 1991 ; references in Rohde et al. 1992). Among the major groups of parasitic Platyhelminthes (Neodermata sensu Ehlers 1985), two types of protonephridia can be distinguished, one characterized by cytoplasmic cords along the weir of the flame bulb connected by a septate junction and by a septate junction in the proximal capillaries, * Financially supported by the Australian Research Council and the University of New England Offprint requests to: K. Rohde
laries, and another without such cords or junction. The first type is found in the Trematoda Digenea, Trematoda Aspidogastrea and Monogenea and the second, in the cestodes, including the Amphilinidea and Gyrocotylidea, as well as in Udonella (Rohde et al. 1992). To date, the development of the protonephridia of only one species has been examined with the electron microscope: that of the amphilinid Austramphilina elongata (see Rohde and Watson 1988). In this paper, we examine the developing protonephridia of the cercaria of Philophthalmus sp. Comparison of the trematode (weir with cytoplasmic cords) with the cestode (weir without cytoplasmic cords) will show whether there are fundamental differences in the development of the protonephridia in the two groups. Materials and methods Rediae containing developing and fully developed cercariae of Philophthalmus sp. were dissected out of Pyrazus obeninus snails collected in Deception Bay near Brisbane in March 1987 and then fixed for 1-2 h in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4~ C. Specimens were washed in the same buffer at 4~ C, postfixed in 1% OsO~ in 0.1 M phosphate buffer (pH 7.2) at room temperature for 30 min, and dehydrated in an alcohol series. They were embedded in Spurr's resin and polymerized overnight at 60~ C. Sections measuring 60-70 nm in thickness were stained with alcoholic uranyl acetate and lead citrate and examined under a Jeol 1200 EX electron microscope operating at 60 kV. Results The fully (or almost fully) developed flame bulb and proximal capillary exhibit the structure characteristic of Trematoda and Monogenea (Figs. 1 5, 23): a terminal cell with a nucleus close to the weir and a proximal canal cell possessing internal and external ribs (rods), respectively, that interdigitate to form the filtration apparatus or weir. Ribs are connected by a filtration " m e m b r a n e " . External processes or leptotriches arise from the external ribs, and internal leptotriches arise
369
Figs. 1-5. Fully or almost fully developed flame bulb. Figs. 1, 3. Longitudinal sections through the flame bulb and proximal capillary (p). Note the perikaryon of the terminal cell (t), the rootlets of cilia (r), and the internal leptotriches (i/). Fig. 2. Longitudinal section through the base of the weir. Note the interdigitating external (er) and internal ribs (it), the former arising from the proximal canal cell and the latter, from the terminal cell (t). Fig. 4. Cross
section through the weir. Note the internal and external ribs connected by a "membrane", the cytoplasmic cords connected by a septate junction (s), and the internal and external (e/) leptotriches. c, cilia. Fig. 5. Cross section through the basal bodies (b) and rootlets (r) of cilia forming the flame. Note the hollow rootlets. Scale bars = 1 gm
370
Figs. 6-9. Sections through the flame bulb and excretory ducts of a cercarial embryo. Figs. 6-8. Developing flame bulb. Note the terminal cell (t), the proximal canal cell (p), the septate junction (s) in the wall of a capillary formed by the canal cell and between the terminal and proximal canal cells, the nucleus (n) of the canal
cell, microtubules (rot), mitochondria (m), and the external and internal (i/) leptotriches. Fig. 9. Excretory duct (ed) near the excretory opening just below the tegument (te) and microtubules (mt). Scale bars = 1 gm
371
Figs. 10-15. Semi-serial cross sections through a developing flame bulb. The cross section shown in Fig. 10 is that most distally located. Note the septate junction (s) between the cytoplasmic cords of the proximal canal cell (p), the sheet-like process of the terminal cell (t) extending into the cylinder formed by the proximal canal cell and connected to it by desmosome-like connections ("membranes", me). The sheet-like processes of the terminal cell break
up into the internal ribs (ir), and the cytoplasm of the outer wall of the cylinder formed by the proximal canal cell breaks up between the attachment zones of the "membranes" (large arrowheads') opposite the internal ribs. On the right of the section, internal and external ribs are arranged in a single row. n, nucleus. Scale bars = 1 pm
372
Figs. 16--22. Sections through a developing flame bulb and excretory duct. Figs. 16-18. Semi-serial oblique sections through the developing flame bulb. Note the terminal (t) and proximal canal cells (p) attached to each other by numerous desmosome-like connections. Also note the basal body (b) in the cytoplasm of the terminal cell, some distance from the base of the flame. Fig. 19. Longitudinal section through an excretory pore (ep) and duct at-
tached by septate junctions (s) to surrounding tissue. Fig. 20. Cross section through the excretory duct. Note the septate junction (s) and numerous branched and looping lamellae extending into the lumen of the duct. Figs. 21-22. Excretory duct (ed) nucleus (n) excretory pore (ep). Note the septate junction around the terminal part of the duct and the lamellae in the duct. Scale bars = 1 pm
373
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O
Fig. 23A-C. Diagrams of a fully developed flame bulb. A Longitudinal section. B Cross section through the proximal capillary. C Cross section through the weir (filtration apparatus), c, cilium; cc, cytoplasmic cord; el, external leptotrich; er, external rib; il, internal leptotrich; internal rib; l, lamella; me, "membrane" ; n, nucleus; p, proximal canal cell; s, septate junction; t, terminal cell; v, vesicle. Scale bar = 1 lain
from the internal ribs and basal cytoplasm of the terminal cell near the basal bodies of the cilia forming the flame. The basal parts of the cilia are separated by narrow spaces, but they remain in tight contact along most of their length, forming a functional whole. The central pairs of microtubules are identically oriented in all cilia (Fig. 4). The cilia have cross-striated rootlets that are hollow along part of their length (Fig. 5). Cytoplasmic cords connected by a septate junction extend along the weir (Fig. 4). The internal surface of the proximal capillary is enlarged by numerous lamella-like projections (Fig. 1).
Whereas the nucleus of the proximal canal cell in fully developed protonephridia is located at a great distance from the terminal cell, it is very close to it in developing protonephridia (Fig. 8), indicating that the proximal canal cell undergoes considerable stretching during development. Furthermore, whereas the perikaryon of the terminal cell, including most of its cytoplasm, is located basal to the flame (seen in longitudinal sections of at least six terminal cells (Figs. 1, 3) in fully developed protonephridia, the terminal cell body in the developing flame bulb extends around much of the flame (observed in ten cells; Figs. 7, 8, 24). During development, processes of the proximal canal cell maintain contact with the terminal cell and probably with each other via a number of septate junctions (Figs. 6, 7), and microtubules extend in a predominantly longitudinal direction in both the proximal canal and the terminal cells (Fig. 6). Sheet-like outgrowths of the terminal cell are externally surrounded by cytoplasm of the proximal canal cell (Figs. 6, 7, 10M 8), which contains a septate junction and is connected to the outgrowths of the terminal cell by numerous desmosome-like structures with thick, electron-dense ends in the cytoplasm of both the proximal canal cell and the terminal cell (Figs. 7, 10-18). Observations of several developing terminal cells showed that during development, the cytoplasmic sheets of the terminal cell break up into longitudinal ribs. The external cytoplasm between the ends of two desmosomelike structures from one internal rib becomes less dense and finally disintegrates, leaving the external ribs connected to the internal ones by a " m e m b r a n e " of apparently extracellular matrix (Figs. 10-15, 24A). Irregular outgrowths of the internal ribs and cytoplasmic sheets represent the internal leptotriches (Figs. 7, 14). Internal and external ribs are sometimes arranged in a single row (Figs. 11M 5, 24A), although distinctly alternating external and internal ribs are evident in fully developed flame bulbs (Figs. 4, 23). Occasionally, basal bodies are seen in the cytoplasm of the terminal cell at some distance from the base of the flame (Fig. 18). Basal bodies and ciliary rootlets of lateral (non-terminal) flames are present in the (proximal?) canal (Fig. 6). Lamellae were observed in the developing proximal canal (Fig. 6). We did not examine whether more than one distal canal cell is present. However, the perikaryon of one distal canal cell is located at some distance from the excretory pore (Fig. 21). The distal excretory duct in the developing cercaria contains numerous branching and looping lamellae, leaving a reticular lumen (Figs. 9, 1922), and a septate junction or junctions (Figs. 19-22). The terminal part of the duct is connected to the surrounding tegument by a septate junction (Figs. 19, 22), and several microtubules extend around it in the tegument near the surface (Fig. 9).
Discussion
The fully developed flame bulb of P h i l o p h t h a l m u s corresponds to that of eight genera of Digenea, three genera of Aspidogastrea and nine genera of the Monogenea
374 A
mt
OoO(
O
9
O
o
o
o
Qo t
Polyopisthocotylea and Monopisthocotylea examined (Rohde et al. 1992) in the presence of two cytoplasmic cords continuous with the proximal canal cell and connected by a septate junction. All genera of the taxa examined in this regard also have a septate junction in the proximal capillary and internal surface lamellae in small capillaries. Only one monopisthocotylean monogenean, Anoplodiscus cirrusspiralis, lacks the cytoplasmic cords and a complete septate junction (both in the flame bulb and the proximal capillary). However, the presence of junctions in larger capillaries and of a rudimentary junction in the proximal capillaries indicate that a complete junction has been secondarily lost. Thus, the features mentioned can be considered to be primary characteristics of Trematoda/Monogenea. On the other hand, Udonella, the two genera of Amphilinidea, one genus of Gyrocotylidea, and seven genera of the Eucestoda
P
Fig. 24A, B. Diagrams of a developing flame bulb. A Cross section through the developing weir. B Longitudinal section through the developing flame bulb. c, cilium; cc, cytoplasmiccord; er, external rib; it, internal rib; l, lamella; rot, microtubule;p, proximal canal cell; n, nucleus; s, septate junction; t, terminal cell. Scale bars = 1 gm
examined lack cytoplasmic cords and a junction in the flame bulb and their capillaries are without junctions; moreover, all of these parasites except Udonella which has smooth-walled capillaries, possess short microvilli instead of lamellae (Rohde et al. 1992). In the only ultrastructural study of the development of the protonephridial system of a platyhelminth, that of the amphilinid AustramphiIina eIongata, Rohde and Watson (1988) have shown that three branches of a com. pletely closed cytoplasmic cylinder (proximal canal cell) enclose the flames of three terminal cells. Processes of the terminal cells grow into the space between the cilia and cylinder, and external ribs are formed at the points of contact between inner outgrowths and cylinders, being connected to the internal ribs by "membranes". In Philophthalmus, the formation of ribs proceeds in a similar fashion, although the inner outgrowths are dis-
375
tinctly sheet-like and (at least usually) do not occur as thin ribs as in Austramphilina. Most importantly, a seprate junction was present at the earliest stages of development observed in Philophthalmus, whereas it was never seen in the cestode. Its occurrence at a very early stage of development in the latter parasite cannot be excluded, but if it occurs at all, it must be of very short duration. Also, the lamellae in the capillaries characteristic of Trematoda/Monogenea and the short microvilli characteristic of the cestodes occur at quite early stages of development in the two species examined. We did not examine whether more than one flame is enclosed by a single proximal canal cell, which is nevertheless likely in view of the observation that flame cells in cercariae are arranged in bundles of at least two. A septate junction located between the terminal part of the excretory duct near the excretory pore and the tegument, such as that found by us in Philophthalmus, has also been demonstrated to occur in the cercaria of Schistosoma mansoni by Ebrahimzadeh and Kraft (1971). Lamellae in the caudal excretory canal are also present in the cercaria of Cryptocotyle lingua, although the interconnections between them cannot be seen in the published figure (Rees 1977).
Acknowledgements. We wish to thank Mr. P. Garlick for making facilities at the Electron Microscope Unit, University of New England, available to us, Mr. R. Porter for developing and contactprinting the negatives, Mr. Z. Enoch for printing the electron mi-
crographs, and Ms. B. Rochester for typing the manuscript.
References Ebrahimzadeh A, Kraft M (1971) Ultrastrukturelle Untersuchungen zur Anatomie der Cercarien yon Schistosoma mansoni: II. Das Exkretionssystem. Z Parasitenkd 36:265-290 Ehlers U (1985) Das phylogenetische System der Platyhelminthes. G. Fischer, Stuttgart New York Rees GF (1977) The development of the tail and the excretory system in the cercaria of Cryptocotyle lingua (Creplin) [Digenea: Heterophyidae] from Littorina littorea (L.). Proc R Soc Lond [Biol] 195:425~452 Rohde K (1990) Phylogeny of Platyhelminthes, with special reference to parasitic groups. Int J Parasitol 20:979-1007 Rohde K (1991). The evolution of protonephridia of the Platyhelminthes. Hydrobiologia 227 : 3/5 321 Rohde K, Watson NA (1988) Development of the protonephridia of Austramphilina elongata. Parasitol Res 74: 255-261 Rohde K, Watson NA, Roubal FR (/992) Ultrastructure of the protonephridial system of Anoplodiscus cirrusspiralis (Monogenea, Monopisthocotylea). Int J Parasitol (in press)
