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Ultrastructure of Trimastix convexa hollande, an amitochondriate anaerobic flagellate with a previously undescribed organization
Europ. J. Protisto!. 33,121-130 (1997) June 30, 1997
EuropeanJournal of
PROTISTOLOGY
Ultrastructure of Trimastix convexa Hollande, an Amitochondriate Anaerobic Flagellate with a Previously Undescribed Organization Guy Bruqerolle' and David Patterson1Laboratoire de Biologie des Protistes, universite Blaise Pascal de Clermont-Ferrand, Aubiere Cedex, France 2School of Biological Sciences, University of Sydney, Australia
Summary Trimastix conoexa is a free-living flagellate with four flagella and found in anaerobic habitats. The light microscopical appearance resembles that of Percolomonas and Tetramitus and some retortamonads, but the flagellate is shown to have an ultrastructural identity that is distinct from that of other quadriflagellate protists such as the Heterolobosea, retortamonads, diplomonads, oxymonads and trichomonads. The cell has a ventral groove bordered on the left by a microtubular root associated with a striated fibre, and on the right by a microtubular root. There are no other major non-microtubular roots or microtubular roots except a poorly developed microtubular dorsal system. The groove contains a recurrent modified flagellum with a vane reminiscent of retortamonads. The cell contains a Golgi apparatus and has hydrogenosome-like organelles but no mitochondria. On the basis of this information, we are unable to assign this species to any of the genera in which it has been previously described, and consequently classify it as Protista incertae sedis.
Key words: Protozoa; Systematics; Phylogeny; Hydrogenosome.
Introduction In 1952 Grasse [16J briefly described Trimastix conuexa Hollande, a flagellate which they assigned to the genus created by Kent 1880 [17J for Trimastix marina. Grasse reported an organism with four subapically inserting flagella, one of the flagella forming an undulating membrane, an anterior nucleus with a large nucleolus, an axostyle and a parabasal body. Grasse [16J suggested on the basis of this information that the organism should be aligned with the trichomonads. To this © 1997 by Gustav Fischer Verlag
date, almost all trichomonads have been described as parasites, although at least two species, Pseudotrichomonas keilini [9, 23J and Ditrichomonas honigbergii [14J, occur as free-living organisms. Flagellates with four flagella, one of which lies in a ventral groove, are also encountered among the amitochondriate retortamonads and in Heterolobosea [23J, such that the suggestion of an affiliation with the trichomonads on the basis of light-microscopy alone is questionable. Given the value of ultrastructural studies in determining the identities of different types of protists [21, 22J and especially of amitochondriate organisms [8, 9J, we have therefore conducted an ultrastructural study of the organism described as Trimastix conuexa to assess its affinities with other groups of organisms [10J.
Material and Methods This flagellate was living in waste water ponds from a sugar refinery near Clermont-Ferrand. Foul-smelling water was collectedin 2 litre jars and allowed to stand at room temperature. After several hours, samples of water were taken at different depths in the jars. Flagellates were first observed by phase contrast microscopy. Cells were concentrated by low speed centrifugation and fixed in a solution of 1% glutaraldehyde in 0.05M Na-cacodylate buffer pH 7.2for 1 hour. After a brief wash with the buffer they were postfixed with a 1% osmic acid solution in Na-cacodylate buffer. Fixed cells were observed by phase contrast microscopy and photographed (Fig. 2). Fixed cellswere pre-embedded in 1% agar before dehydration in an alcohol series and embedding in Epon 812 resin. Sectionswere stained by uranyl acetate and lead citrate before observed with aJeol1200CX or Zeiss902 electron microscope.
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Observations Light microscopy The samples of waste water from the sugar refinery contained a dense biota of bacteria and predominantly two species of flagellates. Trimastix convexa concen-
trated near the bottom of the jars and a colourless volvocine flagellate (Polytoma) on the surface of the water. When observed by phase contrast microscopy Trimastix convexa has an elongated or ovoid cell body shape which has a hunched appearance in side view (Figs. 1,2). It is anteriorly broad, tapering posteriorly. It has a depression which defines the ventral surface and measures 20 pm long by 8 pm wide. The cell has 4 flagella which insert on the ventral surface subapically and at the anterior end of the ventral groove. All flagella beat freely and are unattached to the cell or to each other along their length to the cell body. Typically, one flagellum is directed forward and two are generally oriented backward (Figs. 1, 2). The fourth flagellum is longer than the others and is situated in the ventral groove and extends beyond the posterior end of the cell. Food (bacteria) appears to be ingested in the ventral groove. The cell contains a large anterior nucleus, many food vacuoles with bacteria and some clear vacuoles.
rf
1 Fig. 1. Representation of Trimastix convexa from light microscope observations (phase contrast). The cell has a ventral groove (vg) containing a ventral flagellum (rf), One flagellum is oriented forward, two others are generally oriented backward and the fourth is recurrent lying in a ventral groove. The anterior pear-shaped nucleus (N) contains a large nucleolus, the Golgi (G) is situated at the anterior dorsal part, food vacuoles (N), clear vacuoles (cV) and dense granules are represented.
Fig. 2. Phase contrast microscope photographs of different Trimastix convexa fixed cells. Notice the ventral groove (arrowheads) with an undulated border (Fig. 2c), the recurrent flagellum (r) (Fig. 2b, c, d, e), the nucleus (n) (Fig. 2e), the 3 anterior flagella (1, 2, 3) (Fig. 2a), two ventrally opposed cells (Fig. 2d) and a more elongated one (Fig. 2f). Bar = 10 pm.
Fig. 3. Transverse sections through cells, showing the ventral groove (g) and the ventral modified flagellum (vF), the nucleus (N) containing the large nucleolus and abundant food vacuoles (V). Bar = 1 pm. Fig. 4. Longitudinal section showing the insertion of the anterior flagella (aF) at the top of the cell, the recurrent modified flagellum (vF) in the ventral groove (g), the pear-shape nucleus (N) and its nucleolus (nl), Golgi place (G), food vacuoles (V), hydrogenosome-like granules (H) and adhering bacteria (B). Bar = 1 pm. Fig.5. Longitudinal section of the posterior region through the ventral groove (g) which curves back into the cell at its posterior end, the recurrent flagellum (F) and one clear vacuole (cV). Bar = 1 pm. Fig.6. Transverse section at the levelof the nucleus showing the ventral groove containing the modified flagellum(vF), the right microtubular fibre (rF) and the left-side microtubular fibre (IF)supporting the margins of the groove (G), the endoplasmic reticulum (Er) surrounding the nucleus (N) with its central nucleolus (nl) and tWO membrane adhering bacteria (B).Bar = 1 pm.
Ultrastructure of Trimastix convexa
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Electron microscopic study Th e overall appearance of the cell is shown in transverse (Figs. 3, 6) and longitudinal (Figs. 4,5) sections of th e cell. Transverse sections of the middle of the cell show the ventral groove (g) within which lies the recurrent flagellum (vF) . The groove extends to the posterior of the cell and curves back a little (Fig . 5). The flagella insert very close to the anterior end of the cell, with a dictyosome of the Golgi apparatus lying between the basal bodies and the anterior lobe of the nucleus (Fig. 4). The nucleus has a prominent nucleolus (Fig. 6). The nucleus may be drawn out at its anterior end (Fig. 4) and is surrounded by endoplasmic reticulum. There are numerous food vacuoles which contain bacteria at different stages of digestion, and among them are empty vacuoles, one of which at least we interpret as being a co ntractile vacuole (cV). Some long rod-shaped bacteria adhere to the membrane inside the ventral depression or on the surface of the cell (Figs. 4, 6).
Basal body and associated fibre arrangement The basal bodies (bb) of the 4 flagella are arranged in two pairs. In the first pair bb1 is oriented on the left side and is opposed by its base to bb2 oriented on th e right side (Figs. 7, 8, 9). In the second pair bb3 gives rise to the recurrent flagellum and is orthogonal to bb4 which is oriented on the left side (Figs. 7, 8, 10). Each basal body has the 9 triplet structure and a cartwheel at the proximal part. The flagella have the classic axonemal 9+2 structure, with a densely contrasted axosome (Figs. 8, 9, 11, 15). As iiIdicated below, the recurrent flagellum has paraxial structures. The flagellar apparatus gives rise to three ribbons of microtubules and one non-microtubular root. A ribbon (rF) arises as 5 microtubules between bb2 and bb3 (Fig. 11) and rapidly enlarges to 30 microtubules (Fig. 12) to create a ribbon which lies beneath the right edge of the ventral groove (Figs. 7, 14, 15). A delicate microfibrillar layer is located ashort distance from the externally directed face of this root near its proximal part (Fig. 12). More posteriorly some tightly linked micro-
Fig. 7. Interpretative diagram showing the organisation of the basal bodies and associated roots of Trimastix conuexa. Bb1 and bb2 are opposed at their bases, bb3 of the ventral flagellum (vF) is inserted at right angle to bb4. Origin and course of the right side microtubular root (rF), of the left side root (IF) and of the dorsal microtubular system (dS) are shown.
tubules of rF lie near the edge of the groove, but some microtubules separate and extend along the inner surface of the groove (Fig. 17). Another root (IF) arises as 4 microtubules between bb3 and bb4 and is directed toward the left side of the groove (Figs. 7, 10). The number of microtubules increases to produce a ribbon of about 10 microtubules which is closely associated with a striated fibre (Figs. 7, 16, 19). Each microtubule of this ribbon can be seen to bear an extension when viewed in transverse section
Figs. 8, 9, 10. Figures showing the arrangement of basal bodies. Bb1 and bb2 are opposed by their base. Bb3 of the recurrent flagellum inserts at right angles to bb4 which is directed to the left side. The origin of the dorsal microtubular system (dS, Fig. 9) and left root (IF, Fig. 10) are visible. Bar = 1 pm. Figs. 11, 12, 14. Sections showing the origin, structure and development of the right root (rF). These microtubules arise between bb2 and bb3 (rF in Fig. 11)and form a ribbon which is associated with a microfibrillarlayer near its origin (arrowheads in Fig. 12).This ribbon extends to support the right side of the groove (rF, Fig. 14).Bar = 1 pm. Figs. 13, 15, 16. Sections showing the origin, structure and development of the left root (IF) and of the dorsal microtubular system (dS). The left root (IF) is composed of several microtubules lined by microfibrillar layers (arrowheads Figs. 11 , 16). A nodule (n) is present between the basal bodies (Figs. 8, 11, 13, 14). The dorsal system (dS) is composed of a series of dorsally oriented microtubules originating at the contact of 2 microtubules oriented backward (Figs. 13,16). Bar = 1 pm.
Ultrastructure of Trimastix convexa
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Ultrastructure of Trimastix convexa
(Fig. 18). This fibre is about 300 nm thick and has a periodicity of 33 nm. Near its origin, the striated fibre is lined by an additional microfibrillar bundle (Figs. 11, 16) arising from a nodule, close to basal bodies 2 and 3 (Figs. 8, 10, 11, 13, 14). The composite left root supports the left margin of the groove (Fig. 16). Dorsally, 2 microtubules directed to the left side of the cell arise close to bb2 (Figs. 7, 9, 13, 16). The two microtubules act as MTOC and give rise to a series of microtubules which underlie the dorsal surface of the cell (Figs. 7, 13, 16). This microtubule system seems to be restricted to the dorsal surface of the cell. The ventral (recurrent) flagellum is modified and possesses two lateral fin-like extensions, the right one being larger than the left one (Figs. 6, 15,20,21). Each lateral extension contains a microfibrillar and striated lamina (Figs. 20,21).
Other cytoplasmic structures There are no mitochondria but the cell contains some dense spherical or dumbbell-shaped granules about 0.5 to 1 pm in diameter (Figs. 22, 23). They are limited by two closely apposed unit membranes and contain a heterogeneous dense matrix. They are often surrounded by endoplasmic reticulum cisternae. Close to basal bodies, but lying dorsally, there is a dictyosome of the Golgi apparatus and this is composed of about 10 cisternae surrounded by vesicles (Fig. 24). There is no striated fibre associated with the dictyosome cisternae.
Discussion The flagellate studied here corresponds well with the account of Trimastix convexa Hollande by Grasse [16]. The type species of the genus Trimastix is Trimastix marina Kent 1880 [17], an organism described from saltwater with decaying vegetation. The organism was
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described as being anteriorly pointed, sac-shaped posteriorly, and with three flagella inserting apically. It resembles the organism described here in having a ventral groove and one recurrent flagellum, but differs in the form of the cell and the number of flagella. Grasse [16] presented information on an organism he called Trimastix marina, although it differed in shape and appeared to have two nuclei. The organism described as Trimastix marina has considerable similarity with species assigned to Elvirea, Dallingeria, and Pleuramastix [16, 24]. The identities of evolutionary lineages can be determined successfully from ultrastructural studies, and accounts of the organization of protistan cells allow organisms to be assigned to such lineages [8, 13, 21, 22, 23]. In having four flagella located at the head of a ventral groove, and in having a recurrent flagellum lying within the groove, Trimastix convexa has similarities with the amitochondriate retortamonads and trichomonads, and with some mitochondriate heteroloboseids (e.g. Tetramitus, Psalteriomonas, Percolomonas). As the electron dense bodies bounded by two membranes may be derived from mitochondria, we will make comparisons with both mitochondriate and amito chondriate lineages of four flagella. Retortamonads are amitochondriate mostly parasitic flagellates characterized by having 4 flagella and a ventral groove [8]. There are two genera (Retortamonas and Chilomastix) both of which have been reported as having free-living representatives [9]. Genera of parasitic species can be distinguished by the arrangement of the inserting basal bodies and cytoskeletal structures [5, 7]. The flagella of Retortamonas insert in well segregated pairs unlike Trimastix. The arrangement in Chilomastix is more similar to that of Trimastix, in that the flagella insert as two adjacent pairs, one located more anteriorly, one posteriorly; and one flagellum from the posterior pair is recurrent. However, the angles of insertion of the basal bodies differ in the two genera. The recurrent flagellum of all three taxa has a flagellar wing,
Fig. 17. Transverse section of the right margin of the ventral groove showing that the right root (rF) is composed of a ribbon of microtubules which are linked by microfibrillar material (arrow). Some microtubules detach and spread along the membrane of the groove (arrowheads), adhering bacteria (B). Bar = 1 pm. Figs. 18, 19. Transverse (Fig. 18) and longitudinal section (Fig. 19) of the left margin of the groove (g). The left root is composed of about 10 microtubules from which thin barbs or flanges emerge (arrow, Fig. 18) and these appear as a striated fibre in longitudinal section (arrow Fig. 19). Bar = 1 pm. Figs. 20, 21. Structure of the recurrent modified flagellum (vF) in longitudinal (Fig. 20) and transverse section (Fig. 21) showing the microfibrillar material inside the 2 fin-like extensions along the axonema (arrows). Bar = 1 pm. Figs. 22, 23. Sections showing the hydrogenosome-like granules (H) limited by two closely apposed membranes (arrow in Fig. 22) and containing a dense matrix; some granules are surrounded by endoplasmic reticulum (Er). Fig. 22, Bar = 1 pm, Fig. 23, Bar = 0.5 pm. Figs.24. The dictyosome of the Golgi (G) occupies a dorsal position with respect to the basal bodies bb l and bb4 and the origin of the right root (rF). Bar = 1 pm.
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in all taxa the four basal bodies give rise to three sets of microtubular roots which support the left and right walls of the ventral groove and microtubules which underlie the cell surface. The retortamonads differ from Trimastix because they do not have dictyosomes, no hydrogenosomes or hydrogenosome-like organelles have been reported, because the right lip of the ventral groove is subtended by a microtubular and microfibrillar band, and not the left lip as in Trimastix. Retortamonas has a supranuclear striated fibre and Chilomastix may have such a fibre; no such fibre has been observed in Trimastix. In both retortamonad genera, there are microfibrillar bands linking the cytostomal roots, and the right microtubular ribbon begins as an arc. These features are not observed in Trimastix. The similarities with trichomonads previously indicated by Grasse [16J are only superficial. Trichomonads have an arrangement of basal bodies and associated fibres in which the recurrent flagellum attaches to the cell surface to form an undulating membrane, and the cytoskeletal elements include an axial axostyle and a superficial pelta. In addition, there is a striated root which links the basal bodies to the dictyosome, and a striated fibre lying under the recurrent flagellum [1, 8, 27J. Trichomonads do not have a cytostorne. In spite of the presence of hydrogenosome-like organelles in Trimastix, the genus studied in this paper cannot be assigned to the trichomonads because of a different arrangement of basal bodies, the absence of the costal and para basal striated fibres and an axostyle, as well as the presence of the ventral groove. The ultrastructural comparison of the basal bodies and associated roots in Trimastix and in the amitochondriate diplomonads such as Enteromonas [6, 8J or the exclusively parasitic oxymonads [8, 11J reveal differences in the insertion of the flagella, the number, orientation and associations of the microtubular roots, and in the absence of dictyosomes. The diplomonad Enteromonas has four basal bodies arranged in pairs in close contact with the nucleus..and the basal body attached roots comprise a supra-nuclear root, an infranuclear root and a cytostomal root. This organization is different from that of Trimastix. Oxymonads such as Monocercomonoides and Polymastix [8, 11J have four flagella arising from two pairs of basal bodies separated by a preaxostylar lamina, there is an axial paracrystalline axostyle but no groove or cytostome. Moreover diplomonads and oxymonads do not have a Golgi apparatus, mitochondria or hydrogenosome-like organelles. The Heterolobosea contain a number of genera with biflagellated and quadriflagellated forms. Among them, the genus Tetramitus is a quadriflagellated member of the Heterolobosea [20, 23J. The best characterised species is Tetramitus rostratus (the type of the genus) an
organism with a clear light-microscopical ami electron microscopical identity [2, 19]. The organism is mitochondriate with four basal bodies inserting in pairs, with two major microtubular roots and one well-developed cross-striated root. A variety of other species with a similar morphology have been assigned to Tetramitus [17, 18, 25, 26J and include Tetramitus descissus and T. pyriformis which have a similar morphology and share several characters with Trimastix convexa. One of the species assigned to Tetramitus was T. cosmopolitus which was subsequently studied by Fenchel & Patterson [15J who showed that while it had certain similarities with Tetramitus, it lacked other features (such as the well-developed striated fibre and the number of microtubular roots). As a result Fenchel and Patterson [15J created a new genus Percolomonas, which they located in the Heterolobosea incertae sedis. Fenchel and Patterson [15J argued that T. salinus, T. descissus, T. pyriformis and T. sulcatus might also be better assigned to Percolomonas. The light and electron microscopic studies indicate that Tetramitus salinus is a percolomonad (Gunderson, pers. comm.) but Tetramitus descissus is very close to Psalteriomonas (Brugerolle, unpublished results). The Heterolobosea also contain Psalteriomonas lanternae and p. vulgaris [3, 4J, the latter species also being referred to Lyromonas vulgaris and placed in its own class [12J even though both.species share a number of features with Tetramitus and other Heterolobosea sensu stricto. The studies of Psalteriomonas suggests that there are four microtubular roots associated with the flagellar system. Despite the similarities between Trimastix and Psalteriomonas in the lack of mitochondria, Psalteriomonas differs from Trimastix in the orientation of the flagella, the insertion of the basal bodies and their length, in the source and direction of the roots, in the number and ultrastructure of roots. In the way the microtubules are deployed around the groove and in the existence of well developed striated fibres, Trimastix is quite different to the mitochondriate and amitochondriate vahlkampfiid amoeboflagellates which have been studied to date (i.e. Psalteriomonas, Tetramitus, Naegleria) and assigned to the Heterolobosea. Although Percolomonas has mitochondria, this genus resembles Trimastix more than the vahlkarnpfiid amoeboflagellates because of the lack of derived characters such as the striated fibre and the presence of three microtubular roots, none the less, there are still considerable differences with regard to the presence of mitochondria and the absence of dietyosomes in Percolomonas, in the orientation of the microtubular roots and their deployment especially with regard to the ventral gutter. Trimastix convexa clearly does not have an ultrastructural organization that is identical to that of the -,
Ultrastructure of Trimastix convexa
well-characterised quadriflagellate lineages of protists. It cannot be assigned to any of these lineages and must therefore be placed at "Protista incertae sedis". We do however note that there are similarities with both the amitochondriate heteroloboseids, and believe that attention to the flagellates from anoxic habitats will help in the clarification of the origins and early diversification of the amito chondriate protists.
Diagnoses Genus Trimastix Kent 1880 [15], Grasse and Hollande 1952 [16], emended Brugerolle and Patterson. Free-living flagellated heterotrophic protist, with four subapically inserting flagella, and ventral groove. Without mitochondria or rhizoplast, but with dictyosome and hydrogenosome-like organelles. Affinities uncertain. With two species, Trimastix convexa and Trimastix marina (the type species). Trimastix convexa Hollande in Grasse 1952 [16] Flagellate heterotrophic protist measuring 20 pm in length and with four anterior flagella. One flagellum lying in a ventral groove and longer than the body. Ventral groove extending all along the ventral side of the body. Two flagella oriented on the left side, one flagellum oriented on the right side and a ventral flagellum directed backwards. Two basal bodies are opposed and the other two insert at right angles. Microtubular roots support the right and left rims of the ventral groove. The left fibre is associated with a striated fibre. There is a microtubular system on the left dorsal side. Golgi close to the basal bodies. No mitochondrion, hydrogenosome-like granules. Nucleus with a large nucleolus. Bacterivorous, anaerobic. Acknowledgement: G. B. acknowledges the University of Sydney for providing facilities for electron microscopy, D.]. P. acknowledges support of the ARC and ABRS.
References Amos W. B., Grimstone A. v., Rothschild L. J., and Allen R. D. (1979): Structure, protein composition and birefringence of the costa: a motile flagellar root fibre in the flagellate Trichomonas.]. Cell Sci. 35, 139-164. 2 Balamuth W, Bradbury P. c., and Schuster F. L. (1983): Ultrastructure of the amoeboflagellate Tetramitus rostratus. ]. Protozool. 30, 445-455. 3 Broers C. A. M., Stumm C. K., Vogels G. D., and Brugerolle G. (1990): Psalteriomonas lanterna gen. nov., sp. nov. a free-living amoeboflagellate isolated from freshwater anaerobic sediments. Europ. ]. Protistol. 25, 369-380. 4 Broers C. A. M., Meijers H. H. M., Symens J. c., Stumm C. K., Vogels G., and Brugerolle G. (1993): Symbiotic association of Psalteriomonas vulgaris n. spec. with Methanobaeterium formicicum. Europ.]. Protistol. 29, 89-105.
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5 Brugerolle G. (1973): Etude ultrastructurale du trophozoite et du kyste chez Ie genre Chilomastix Alexeieff, 1910 (Zoomastigophorea, Retortamonadida Grasse, 1952). ]. Protozool. 20, 574-585. 6 Brugerolle G. (1975): Etude ultrastructurale du genre Enteromonas da Fonseca (Zoomastigophorea) et revision de l'ordre des Diplomonadida Wenyon. ]. Protozool. 22, 468-475. 7 Brugerolle G. (1977): Etude ultrastructurale du genre Retortamonas Grassi 1879 (Zoomastigophorea, Retortamonadida Wenrich 1931). Protistologica 13,233-240. 8 Brugerolle G. (1991): Flagellar and cytoskeletal systems in amitochondrial flagellates: Archamoebae, Metamonada and Parabasala. Protoplasma 164,70-90. 9 Brugerolle G. (1991): Organization of amitochondriate flagellates. In: Patterson D. ]. and Larsen]. (eds.): The biology of free-living heterotrophic flagellates, pp. 133-148. Clarendon Press, Oxford. 10 Brugerolle G. (1995): A free-living amitochondriate flagellate without close relationships with Percolozoa, retortamonad or trichomonad flagellates. Europ. ]. Protistol. 31,410. 11 Brugerolle G. et Joyon L. (1973): Ultrastructure du genre Monocercomonoides (Travis). Zooflagellata, Oxymonadida. Protistologica 9,71-80. 12 Cavalier-Smith T. (1993): Kingdom Protozoa and its 18 phyla. Microbiol. Revs. 57,953-994. 13 Corliss J. O. (1987): Protistan phylogeny and eukaryogenesis. Int. Rev. Cytol. 100, 319-370. 14 Farmer M. (1993): Ultrastructure of Ditrichomonas honigbergii n. g., n. sp. (Parabasalia) and its relationships to amitochondrial protists. ]. Euk. Microbiol. 40, 619-626. 15 Fenchel T. and Patterson D. J. (1986): Percolomonas cosmopolitus (Ruinen) n. gen., a new type of filter feeding flagellate from marine plankton. ]. mar. bioI. U. K. 66, 465-482: 16 Grasse P. P. (1952): Ordre des Trichomonadines. In: Grasse P. P. (ed.): Traite de Zoologie, Vol. 1, Fasc. 1, p. 704. Masson, Paris. 17 Kent W. S. (1880-1882): Manual of the Infusoria: flagellate, ciliate and tentaculiferous Protozoa, British and Foreign, vol. 1, pp. 310-315. 18 Klug G. (1936): Neue oder wenig bekannte Arten der Gattungen Mastigamoeba, Mastigelia, Cercobodo, Tetramitus und Trigonomonas. Arch.Jfotistenk. 87, 97-116. 19 Outka D. E. and Kluss B. C. (1967): The ameba to flagellate transformation in Tetramitus rostratus. II. Microtubular morphogenesis.]. Cell BioI. 35, 323-346. 20 Page F. C. and Blanton R. L. (1985): The Heterolobosea (Sarcodina: Rhizopoda), a new class uniting the Schizopyrenida and the Acrasidae (Acrasida). Protistologica 12, 101-120. 21 Patterson D. ]. (1994): Protozoa, evolution and classification. Progress in Protozoology, Proceedings of the IX International Congress of Protozoology, pp. 1-14. Fischer Verlag, Stuttgart. 22 Patterson D. J. and Brugerolle G. (1988): The ultrastructural identity of Stephanopogon apogon and the relatedness of the genus to other kinds of protists. Europ. J. Protistol. 23, 279-290. 23 Patterson D. ]. and Larsen ]. (1991): The biology of free-living heterotrophic flagellates. Clarendon Press, Oxford.
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24 Patterson D.]. and Zolffel M. (1991): Heterotrophic flagellates of uncertain taxonomic position. In: Patterson D. J. and Larsen J. (cds.): The biology of free-living heterotrophic flagellates, pp. 427-475. Clarendon Press, Oxford. 25 Ruinen]. (1938): Notizen uber Salzflagellaten. II. Ober die Verbreitung der Salzflagellaten. Arch. Protistenk. 90, 200-258. 26 Skuja H. (1956): Taxonomische und biologische Studien uber das Phytoplankton schwcdischer Binnengewasser, Nova acta Rcggiae Societatis societarum upsalinensis 16, pp.404.
27 Viscogliosi E. and Brugerolle G. (1994): Striated fibrcs in trichomonads: costa proteins represent a new class of proteins forming striated roots. Cell Motil. Cytoske!. 29, 82-93.
Address of correspondence: G. Brugerollc, Laboratoirc dc Biologic dcs Protistcs, URA CNRS 1944, U nivcrsite Blaise Pascal dc Clcrmont-Ferrand, F 63177, Aubiere Cedcx, France.
EuropeanJournal of
PROTISTOLOGY
Ultrastructure of Trimastix convexa Hollande, an Amitochondriate Anaerobic Flagellate with a Previously Undescribed Organization Guy Bruqerolle' and David Patterson1Laboratoire de Biologie des Protistes, universite Blaise Pascal de Clermont-Ferrand, Aubiere Cedex, France 2School of Biological Sciences, University of Sydney, Australia
Summary Trimastix conoexa is a free-living flagellate with four flagella and found in anaerobic habitats. The light microscopical appearance resembles that of Percolomonas and Tetramitus and some retortamonads, but the flagellate is shown to have an ultrastructural identity that is distinct from that of other quadriflagellate protists such as the Heterolobosea, retortamonads, diplomonads, oxymonads and trichomonads. The cell has a ventral groove bordered on the left by a microtubular root associated with a striated fibre, and on the right by a microtubular root. There are no other major non-microtubular roots or microtubular roots except a poorly developed microtubular dorsal system. The groove contains a recurrent modified flagellum with a vane reminiscent of retortamonads. The cell contains a Golgi apparatus and has hydrogenosome-like organelles but no mitochondria. On the basis of this information, we are unable to assign this species to any of the genera in which it has been previously described, and consequently classify it as Protista incertae sedis.
Key words: Protozoa; Systematics; Phylogeny; Hydrogenosome.
Introduction In 1952 Grasse [16J briefly described Trimastix conuexa Hollande, a flagellate which they assigned to the genus created by Kent 1880 [17J for Trimastix marina. Grasse reported an organism with four subapically inserting flagella, one of the flagella forming an undulating membrane, an anterior nucleus with a large nucleolus, an axostyle and a parabasal body. Grasse [16J suggested on the basis of this information that the organism should be aligned with the trichomonads. To this © 1997 by Gustav Fischer Verlag
date, almost all trichomonads have been described as parasites, although at least two species, Pseudotrichomonas keilini [9, 23J and Ditrichomonas honigbergii [14J, occur as free-living organisms. Flagellates with four flagella, one of which lies in a ventral groove, are also encountered among the amitochondriate retortamonads and in Heterolobosea [23J, such that the suggestion of an affiliation with the trichomonads on the basis of light-microscopy alone is questionable. Given the value of ultrastructural studies in determining the identities of different types of protists [21, 22J and especially of amitochondriate organisms [8, 9J, we have therefore conducted an ultrastructural study of the organism described as Trimastix conuexa to assess its affinities with other groups of organisms [10J.
Material and Methods This flagellate was living in waste water ponds from a sugar refinery near Clermont-Ferrand. Foul-smelling water was collectedin 2 litre jars and allowed to stand at room temperature. After several hours, samples of water were taken at different depths in the jars. Flagellates were first observed by phase contrast microscopy. Cells were concentrated by low speed centrifugation and fixed in a solution of 1% glutaraldehyde in 0.05M Na-cacodylate buffer pH 7.2for 1 hour. After a brief wash with the buffer they were postfixed with a 1% osmic acid solution in Na-cacodylate buffer. Fixed cells were observed by phase contrast microscopy and photographed (Fig. 2). Fixed cellswere pre-embedded in 1% agar before dehydration in an alcohol series and embedding in Epon 812 resin. Sectionswere stained by uranyl acetate and lead citrate before observed with aJeol1200CX or Zeiss902 electron microscope.
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Observations Light microscopy The samples of waste water from the sugar refinery contained a dense biota of bacteria and predominantly two species of flagellates. Trimastix convexa concen-
trated near the bottom of the jars and a colourless volvocine flagellate (Polytoma) on the surface of the water. When observed by phase contrast microscopy Trimastix convexa has an elongated or ovoid cell body shape which has a hunched appearance in side view (Figs. 1,2). It is anteriorly broad, tapering posteriorly. It has a depression which defines the ventral surface and measures 20 pm long by 8 pm wide. The cell has 4 flagella which insert on the ventral surface subapically and at the anterior end of the ventral groove. All flagella beat freely and are unattached to the cell or to each other along their length to the cell body. Typically, one flagellum is directed forward and two are generally oriented backward (Figs. 1, 2). The fourth flagellum is longer than the others and is situated in the ventral groove and extends beyond the posterior end of the cell. Food (bacteria) appears to be ingested in the ventral groove. The cell contains a large anterior nucleus, many food vacuoles with bacteria and some clear vacuoles.
rf
1 Fig. 1. Representation of Trimastix convexa from light microscope observations (phase contrast). The cell has a ventral groove (vg) containing a ventral flagellum (rf), One flagellum is oriented forward, two others are generally oriented backward and the fourth is recurrent lying in a ventral groove. The anterior pear-shaped nucleus (N) contains a large nucleolus, the Golgi (G) is situated at the anterior dorsal part, food vacuoles (N), clear vacuoles (cV) and dense granules are represented.
Fig. 2. Phase contrast microscope photographs of different Trimastix convexa fixed cells. Notice the ventral groove (arrowheads) with an undulated border (Fig. 2c), the recurrent flagellum (r) (Fig. 2b, c, d, e), the nucleus (n) (Fig. 2e), the 3 anterior flagella (1, 2, 3) (Fig. 2a), two ventrally opposed cells (Fig. 2d) and a more elongated one (Fig. 2f). Bar = 10 pm.
Fig. 3. Transverse sections through cells, showing the ventral groove (g) and the ventral modified flagellum (vF), the nucleus (N) containing the large nucleolus and abundant food vacuoles (V). Bar = 1 pm. Fig. 4. Longitudinal section showing the insertion of the anterior flagella (aF) at the top of the cell, the recurrent modified flagellum (vF) in the ventral groove (g), the pear-shape nucleus (N) and its nucleolus (nl), Golgi place (G), food vacuoles (V), hydrogenosome-like granules (H) and adhering bacteria (B). Bar = 1 pm. Fig.5. Longitudinal section of the posterior region through the ventral groove (g) which curves back into the cell at its posterior end, the recurrent flagellum (F) and one clear vacuole (cV). Bar = 1 pm. Fig.6. Transverse section at the levelof the nucleus showing the ventral groove containing the modified flagellum(vF), the right microtubular fibre (rF) and the left-side microtubular fibre (IF)supporting the margins of the groove (G), the endoplasmic reticulum (Er) surrounding the nucleus (N) with its central nucleolus (nl) and tWO membrane adhering bacteria (B).Bar = 1 pm.
Ultrastructure of Trimastix convexa
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Electron microscopic study Th e overall appearance of the cell is shown in transverse (Figs. 3, 6) and longitudinal (Figs. 4,5) sections of th e cell. Transverse sections of the middle of the cell show the ventral groove (g) within which lies the recurrent flagellum (vF) . The groove extends to the posterior of the cell and curves back a little (Fig . 5). The flagella insert very close to the anterior end of the cell, with a dictyosome of the Golgi apparatus lying between the basal bodies and the anterior lobe of the nucleus (Fig. 4). The nucleus has a prominent nucleolus (Fig. 6). The nucleus may be drawn out at its anterior end (Fig. 4) and is surrounded by endoplasmic reticulum. There are numerous food vacuoles which contain bacteria at different stages of digestion, and among them are empty vacuoles, one of which at least we interpret as being a co ntractile vacuole (cV). Some long rod-shaped bacteria adhere to the membrane inside the ventral depression or on the surface of the cell (Figs. 4, 6).
Basal body and associated fibre arrangement The basal bodies (bb) of the 4 flagella are arranged in two pairs. In the first pair bb1 is oriented on the left side and is opposed by its base to bb2 oriented on th e right side (Figs. 7, 8, 9). In the second pair bb3 gives rise to the recurrent flagellum and is orthogonal to bb4 which is oriented on the left side (Figs. 7, 8, 10). Each basal body has the 9 triplet structure and a cartwheel at the proximal part. The flagella have the classic axonemal 9+2 structure, with a densely contrasted axosome (Figs. 8, 9, 11, 15). As iiIdicated below, the recurrent flagellum has paraxial structures. The flagellar apparatus gives rise to three ribbons of microtubules and one non-microtubular root. A ribbon (rF) arises as 5 microtubules between bb2 and bb3 (Fig. 11) and rapidly enlarges to 30 microtubules (Fig. 12) to create a ribbon which lies beneath the right edge of the ventral groove (Figs. 7, 14, 15). A delicate microfibrillar layer is located ashort distance from the externally directed face of this root near its proximal part (Fig. 12). More posteriorly some tightly linked micro-
Fig. 7. Interpretative diagram showing the organisation of the basal bodies and associated roots of Trimastix conuexa. Bb1 and bb2 are opposed at their bases, bb3 of the ventral flagellum (vF) is inserted at right angle to bb4. Origin and course of the right side microtubular root (rF), of the left side root (IF) and of the dorsal microtubular system (dS) are shown.
tubules of rF lie near the edge of the groove, but some microtubules separate and extend along the inner surface of the groove (Fig. 17). Another root (IF) arises as 4 microtubules between bb3 and bb4 and is directed toward the left side of the groove (Figs. 7, 10). The number of microtubules increases to produce a ribbon of about 10 microtubules which is closely associated with a striated fibre (Figs. 7, 16, 19). Each microtubule of this ribbon can be seen to bear an extension when viewed in transverse section
Figs. 8, 9, 10. Figures showing the arrangement of basal bodies. Bb1 and bb2 are opposed by their base. Bb3 of the recurrent flagellum inserts at right angles to bb4 which is directed to the left side. The origin of the dorsal microtubular system (dS, Fig. 9) and left root (IF, Fig. 10) are visible. Bar = 1 pm. Figs. 11, 12, 14. Sections showing the origin, structure and development of the right root (rF). These microtubules arise between bb2 and bb3 (rF in Fig. 11)and form a ribbon which is associated with a microfibrillarlayer near its origin (arrowheads in Fig. 12).This ribbon extends to support the right side of the groove (rF, Fig. 14).Bar = 1 pm. Figs. 13, 15, 16. Sections showing the origin, structure and development of the left root (IF) and of the dorsal microtubular system (dS). The left root (IF) is composed of several microtubules lined by microfibrillar layers (arrowheads Figs. 11 , 16). A nodule (n) is present between the basal bodies (Figs. 8, 11, 13, 14). The dorsal system (dS) is composed of a series of dorsally oriented microtubules originating at the contact of 2 microtubules oriented backward (Figs. 13,16). Bar = 1 pm.
Ultrastructure of Trimastix convexa
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Ultrastructure of Trimastix convexa
(Fig. 18). This fibre is about 300 nm thick and has a periodicity of 33 nm. Near its origin, the striated fibre is lined by an additional microfibrillar bundle (Figs. 11, 16) arising from a nodule, close to basal bodies 2 and 3 (Figs. 8, 10, 11, 13, 14). The composite left root supports the left margin of the groove (Fig. 16). Dorsally, 2 microtubules directed to the left side of the cell arise close to bb2 (Figs. 7, 9, 13, 16). The two microtubules act as MTOC and give rise to a series of microtubules which underlie the dorsal surface of the cell (Figs. 7, 13, 16). This microtubule system seems to be restricted to the dorsal surface of the cell. The ventral (recurrent) flagellum is modified and possesses two lateral fin-like extensions, the right one being larger than the left one (Figs. 6, 15,20,21). Each lateral extension contains a microfibrillar and striated lamina (Figs. 20,21).
Other cytoplasmic structures There are no mitochondria but the cell contains some dense spherical or dumbbell-shaped granules about 0.5 to 1 pm in diameter (Figs. 22, 23). They are limited by two closely apposed unit membranes and contain a heterogeneous dense matrix. They are often surrounded by endoplasmic reticulum cisternae. Close to basal bodies, but lying dorsally, there is a dictyosome of the Golgi apparatus and this is composed of about 10 cisternae surrounded by vesicles (Fig. 24). There is no striated fibre associated with the dictyosome cisternae.
Discussion The flagellate studied here corresponds well with the account of Trimastix convexa Hollande by Grasse [16]. The type species of the genus Trimastix is Trimastix marina Kent 1880 [17], an organism described from saltwater with decaying vegetation. The organism was
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described as being anteriorly pointed, sac-shaped posteriorly, and with three flagella inserting apically. It resembles the organism described here in having a ventral groove and one recurrent flagellum, but differs in the form of the cell and the number of flagella. Grasse [16] presented information on an organism he called Trimastix marina, although it differed in shape and appeared to have two nuclei. The organism described as Trimastix marina has considerable similarity with species assigned to Elvirea, Dallingeria, and Pleuramastix [16, 24]. The identities of evolutionary lineages can be determined successfully from ultrastructural studies, and accounts of the organization of protistan cells allow organisms to be assigned to such lineages [8, 13, 21, 22, 23]. In having four flagella located at the head of a ventral groove, and in having a recurrent flagellum lying within the groove, Trimastix convexa has similarities with the amitochondriate retortamonads and trichomonads, and with some mitochondriate heteroloboseids (e.g. Tetramitus, Psalteriomonas, Percolomonas). As the electron dense bodies bounded by two membranes may be derived from mitochondria, we will make comparisons with both mitochondriate and amito chondriate lineages of four flagella. Retortamonads are amitochondriate mostly parasitic flagellates characterized by having 4 flagella and a ventral groove [8]. There are two genera (Retortamonas and Chilomastix) both of which have been reported as having free-living representatives [9]. Genera of parasitic species can be distinguished by the arrangement of the inserting basal bodies and cytoskeletal structures [5, 7]. The flagella of Retortamonas insert in well segregated pairs unlike Trimastix. The arrangement in Chilomastix is more similar to that of Trimastix, in that the flagella insert as two adjacent pairs, one located more anteriorly, one posteriorly; and one flagellum from the posterior pair is recurrent. However, the angles of insertion of the basal bodies differ in the two genera. The recurrent flagellum of all three taxa has a flagellar wing,
Fig. 17. Transverse section of the right margin of the ventral groove showing that the right root (rF) is composed of a ribbon of microtubules which are linked by microfibrillar material (arrow). Some microtubules detach and spread along the membrane of the groove (arrowheads), adhering bacteria (B). Bar = 1 pm. Figs. 18, 19. Transverse (Fig. 18) and longitudinal section (Fig. 19) of the left margin of the groove (g). The left root is composed of about 10 microtubules from which thin barbs or flanges emerge (arrow, Fig. 18) and these appear as a striated fibre in longitudinal section (arrow Fig. 19). Bar = 1 pm. Figs. 20, 21. Structure of the recurrent modified flagellum (vF) in longitudinal (Fig. 20) and transverse section (Fig. 21) showing the microfibrillar material inside the 2 fin-like extensions along the axonema (arrows). Bar = 1 pm. Figs. 22, 23. Sections showing the hydrogenosome-like granules (H) limited by two closely apposed membranes (arrow in Fig. 22) and containing a dense matrix; some granules are surrounded by endoplasmic reticulum (Er). Fig. 22, Bar = 1 pm, Fig. 23, Bar = 0.5 pm. Figs.24. The dictyosome of the Golgi (G) occupies a dorsal position with respect to the basal bodies bb l and bb4 and the origin of the right root (rF). Bar = 1 pm.
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in all taxa the four basal bodies give rise to three sets of microtubular roots which support the left and right walls of the ventral groove and microtubules which underlie the cell surface. The retortamonads differ from Trimastix because they do not have dictyosomes, no hydrogenosomes or hydrogenosome-like organelles have been reported, because the right lip of the ventral groove is subtended by a microtubular and microfibrillar band, and not the left lip as in Trimastix. Retortamonas has a supranuclear striated fibre and Chilomastix may have such a fibre; no such fibre has been observed in Trimastix. In both retortamonad genera, there are microfibrillar bands linking the cytostomal roots, and the right microtubular ribbon begins as an arc. These features are not observed in Trimastix. The similarities with trichomonads previously indicated by Grasse [16J are only superficial. Trichomonads have an arrangement of basal bodies and associated fibres in which the recurrent flagellum attaches to the cell surface to form an undulating membrane, and the cytoskeletal elements include an axial axostyle and a superficial pelta. In addition, there is a striated root which links the basal bodies to the dictyosome, and a striated fibre lying under the recurrent flagellum [1, 8, 27J. Trichomonads do not have a cytostorne. In spite of the presence of hydrogenosome-like organelles in Trimastix, the genus studied in this paper cannot be assigned to the trichomonads because of a different arrangement of basal bodies, the absence of the costal and para basal striated fibres and an axostyle, as well as the presence of the ventral groove. The ultrastructural comparison of the basal bodies and associated roots in Trimastix and in the amitochondriate diplomonads such as Enteromonas [6, 8J or the exclusively parasitic oxymonads [8, 11J reveal differences in the insertion of the flagella, the number, orientation and associations of the microtubular roots, and in the absence of dictyosomes. The diplomonad Enteromonas has four basal bodies arranged in pairs in close contact with the nucleus..and the basal body attached roots comprise a supra-nuclear root, an infranuclear root and a cytostomal root. This organization is different from that of Trimastix. Oxymonads such as Monocercomonoides and Polymastix [8, 11J have four flagella arising from two pairs of basal bodies separated by a preaxostylar lamina, there is an axial paracrystalline axostyle but no groove or cytostome. Moreover diplomonads and oxymonads do not have a Golgi apparatus, mitochondria or hydrogenosome-like organelles. The Heterolobosea contain a number of genera with biflagellated and quadriflagellated forms. Among them, the genus Tetramitus is a quadriflagellated member of the Heterolobosea [20, 23J. The best characterised species is Tetramitus rostratus (the type of the genus) an
organism with a clear light-microscopical ami electron microscopical identity [2, 19]. The organism is mitochondriate with four basal bodies inserting in pairs, with two major microtubular roots and one well-developed cross-striated root. A variety of other species with a similar morphology have been assigned to Tetramitus [17, 18, 25, 26J and include Tetramitus descissus and T. pyriformis which have a similar morphology and share several characters with Trimastix convexa. One of the species assigned to Tetramitus was T. cosmopolitus which was subsequently studied by Fenchel & Patterson [15J who showed that while it had certain similarities with Tetramitus, it lacked other features (such as the well-developed striated fibre and the number of microtubular roots). As a result Fenchel and Patterson [15J created a new genus Percolomonas, which they located in the Heterolobosea incertae sedis. Fenchel and Patterson [15J argued that T. salinus, T. descissus, T. pyriformis and T. sulcatus might also be better assigned to Percolomonas. The light and electron microscopic studies indicate that Tetramitus salinus is a percolomonad (Gunderson, pers. comm.) but Tetramitus descissus is very close to Psalteriomonas (Brugerolle, unpublished results). The Heterolobosea also contain Psalteriomonas lanternae and p. vulgaris [3, 4J, the latter species also being referred to Lyromonas vulgaris and placed in its own class [12J even though both.species share a number of features with Tetramitus and other Heterolobosea sensu stricto. The studies of Psalteriomonas suggests that there are four microtubular roots associated with the flagellar system. Despite the similarities between Trimastix and Psalteriomonas in the lack of mitochondria, Psalteriomonas differs from Trimastix in the orientation of the flagella, the insertion of the basal bodies and their length, in the source and direction of the roots, in the number and ultrastructure of roots. In the way the microtubules are deployed around the groove and in the existence of well developed striated fibres, Trimastix is quite different to the mitochondriate and amitochondriate vahlkampfiid amoeboflagellates which have been studied to date (i.e. Psalteriomonas, Tetramitus, Naegleria) and assigned to the Heterolobosea. Although Percolomonas has mitochondria, this genus resembles Trimastix more than the vahlkarnpfiid amoeboflagellates because of the lack of derived characters such as the striated fibre and the presence of three microtubular roots, none the less, there are still considerable differences with regard to the presence of mitochondria and the absence of dietyosomes in Percolomonas, in the orientation of the microtubular roots and their deployment especially with regard to the ventral gutter. Trimastix convexa clearly does not have an ultrastructural organization that is identical to that of the -,
Ultrastructure of Trimastix convexa
well-characterised quadriflagellate lineages of protists. It cannot be assigned to any of these lineages and must therefore be placed at "Protista incertae sedis". We do however note that there are similarities with both the amitochondriate heteroloboseids, and believe that attention to the flagellates from anoxic habitats will help in the clarification of the origins and early diversification of the amito chondriate protists.
Diagnoses Genus Trimastix Kent 1880 [15], Grasse and Hollande 1952 [16], emended Brugerolle and Patterson. Free-living flagellated heterotrophic protist, with four subapically inserting flagella, and ventral groove. Without mitochondria or rhizoplast, but with dictyosome and hydrogenosome-like organelles. Affinities uncertain. With two species, Trimastix convexa and Trimastix marina (the type species). Trimastix convexa Hollande in Grasse 1952 [16] Flagellate heterotrophic protist measuring 20 pm in length and with four anterior flagella. One flagellum lying in a ventral groove and longer than the body. Ventral groove extending all along the ventral side of the body. Two flagella oriented on the left side, one flagellum oriented on the right side and a ventral flagellum directed backwards. Two basal bodies are opposed and the other two insert at right angles. Microtubular roots support the right and left rims of the ventral groove. The left fibre is associated with a striated fibre. There is a microtubular system on the left dorsal side. Golgi close to the basal bodies. No mitochondrion, hydrogenosome-like granules. Nucleus with a large nucleolus. Bacterivorous, anaerobic. Acknowledgement: G. B. acknowledges the University of Sydney for providing facilities for electron microscopy, D.]. P. acknowledges support of the ARC and ABRS.
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5 Brugerolle G. (1973): Etude ultrastructurale du trophozoite et du kyste chez Ie genre Chilomastix Alexeieff, 1910 (Zoomastigophorea, Retortamonadida Grasse, 1952). ]. Protozool. 20, 574-585. 6 Brugerolle G. (1975): Etude ultrastructurale du genre Enteromonas da Fonseca (Zoomastigophorea) et revision de l'ordre des Diplomonadida Wenyon. ]. Protozool. 22, 468-475. 7 Brugerolle G. (1977): Etude ultrastructurale du genre Retortamonas Grassi 1879 (Zoomastigophorea, Retortamonadida Wenrich 1931). Protistologica 13,233-240. 8 Brugerolle G. (1991): Flagellar and cytoskeletal systems in amitochondrial flagellates: Archamoebae, Metamonada and Parabasala. Protoplasma 164,70-90. 9 Brugerolle G. (1991): Organization of amitochondriate flagellates. In: Patterson D. ]. and Larsen]. (eds.): The biology of free-living heterotrophic flagellates, pp. 133-148. Clarendon Press, Oxford. 10 Brugerolle G. (1995): A free-living amitochondriate flagellate without close relationships with Percolozoa, retortamonad or trichomonad flagellates. Europ. ]. Protistol. 31,410. 11 Brugerolle G. et Joyon L. (1973): Ultrastructure du genre Monocercomonoides (Travis). Zooflagellata, Oxymonadida. Protistologica 9,71-80. 12 Cavalier-Smith T. (1993): Kingdom Protozoa and its 18 phyla. Microbiol. Revs. 57,953-994. 13 Corliss J. O. (1987): Protistan phylogeny and eukaryogenesis. Int. Rev. Cytol. 100, 319-370. 14 Farmer M. (1993): Ultrastructure of Ditrichomonas honigbergii n. g., n. sp. (Parabasalia) and its relationships to amitochondrial protists. ]. Euk. Microbiol. 40, 619-626. 15 Fenchel T. and Patterson D. J. (1986): Percolomonas cosmopolitus (Ruinen) n. gen., a new type of filter feeding flagellate from marine plankton. ]. mar. bioI. U. K. 66, 465-482: 16 Grasse P. P. (1952): Ordre des Trichomonadines. In: Grasse P. P. (ed.): Traite de Zoologie, Vol. 1, Fasc. 1, p. 704. Masson, Paris. 17 Kent W. S. (1880-1882): Manual of the Infusoria: flagellate, ciliate and tentaculiferous Protozoa, British and Foreign, vol. 1, pp. 310-315. 18 Klug G. (1936): Neue oder wenig bekannte Arten der Gattungen Mastigamoeba, Mastigelia, Cercobodo, Tetramitus und Trigonomonas. Arch.Jfotistenk. 87, 97-116. 19 Outka D. E. and Kluss B. C. (1967): The ameba to flagellate transformation in Tetramitus rostratus. II. Microtubular morphogenesis.]. Cell BioI. 35, 323-346. 20 Page F. C. and Blanton R. L. (1985): The Heterolobosea (Sarcodina: Rhizopoda), a new class uniting the Schizopyrenida and the Acrasidae (Acrasida). Protistologica 12, 101-120. 21 Patterson D. ]. (1994): Protozoa, evolution and classification. Progress in Protozoology, Proceedings of the IX International Congress of Protozoology, pp. 1-14. Fischer Verlag, Stuttgart. 22 Patterson D. J. and Brugerolle G. (1988): The ultrastructural identity of Stephanopogon apogon and the relatedness of the genus to other kinds of protists. Europ. J. Protistol. 23, 279-290. 23 Patterson D. ]. and Larsen ]. (1991): The biology of free-living heterotrophic flagellates. Clarendon Press, Oxford.
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Address of correspondence: G. Brugerollc, Laboratoirc dc Biologic dcs Protistcs, URA CNRS 1944, U nivcrsite Blaise Pascal dc Clcrmont-Ferrand, F 63177, Aubiere Cedcx, France.