- Email: [email protected]
Ultrastructural study of Pentatrichomonoides sp., a trichomonad flagellate from Mastotermes darwiniensis
Europ.]. Protistol. 30, 372-378 (1994) November 25, 1994
Ultrastructural Study of Pentatrichomonoides sp., a Trichomonad Flagellate from Mastotermes darwiniensis Guy Brugerolle1, Alfred Breunig 2 and Helmut K6nig 2 1 Laboratoire de Biologie des Protistes, Universite Blaise Pascal de Clermont-Ferrand, Aubiere, France 2 Angewandte Mikrobiologie, Universitat Ulm, VIm, Germany
SUMMARY The features of one trichomonad flagellate found in Mastotermes correspond to that of Pentatrichomonoides Kirby.The5 basal bodiesof the anterior flagella arearranged like in other trichomonads and particularly like in Hexamastix. The herring bone striation pattern of the costa and the lamellar shape of the UM are very closely related to those of the genus Trichomitopsis and, more generally said, to the Trichomonadinae genera: Trichomonas, Tetratrichomonas and Pentatrichomonas. Asignificant modificationconcernsthe microtubules ofthe axostylewhich do not forman axialhollow tube buta rowof microtubulesunderlying the plasma membrane except in the UM region which is the endocytotic area for the cell. Alsothe preaxostylar fibres subdivide into thin lamellae never seen in a trichomonad.
Introduction The parabasalid group represents a broad evolutionary lineage comprising the small trichomonads and the large hypermastigids. The extent and composition of the lineage are known from the studies of former authors using classical cytology and light microscopy [7, 9, 11, 12]. Many genera of trichomonads have been studied by modern techniques such as electron microscopy [3, 4, 8, 10] but those which compose the complex and varied fauna of the termites are difficult to collect and to screen and many genera have not been investigated. The completion of these comparative studies is necessary to establish the phylogeny of this group [19]. In addition it is very useful to study the biochemical and immunological diversification of the cytoskeleton in these flagellates [5, 17, 18].
We have had the opportunity to examine the fauna of the most primitive termite Mastotermes darwiniensis and to investigate a large trichomon ad corresponding to the genus Pentatrichomonoides. This genus and the species P. seroa were very precisely described by Kirby 1931 [11] in Cryptotermes dudley Banks and C. longieollis Banks of Panama and had been assigned to a new subfamily Penratrichomonoidinae by Honigberg in 1963 [9]. Though not reported by Sutherland 1933 [15] in Australian termites and by Yamin 1982 [20], a similar species has been recognized by Cleveland in Mastotermes darwiniensis (reported by Honigberg 1963 [9]) and a second one in Cryptotermes lamaniensis by Grasse 1952 [7]. Our study is in agreement to that of Kirby [11] and discloses some cytological peculiarities interesting for the compar ative cytology of trichomonad s and for their evolution [19].
Fig. 1. Light microscopicviews of Pentatrichomonoides sp.Phasecontrast photographofa slender cell(Fig. 1a)and Giemsastainingof a massive cell (Fig. 1b).The anterior flagella (af), theundulatingmembrane (urn) andthe nucleus (N) are recognizable. Bar = 10 !tm.Figs. 2, 3. Scanning EMviews of a massive form (Fig. 2) and a slender form (Fig. 3), which is spiralled and truncated at the posterior end. The 5 anterior free flagella and the undulating membrane (UM) are distinguishable. Bar = 5 urn. - Fig. 4. Enlarged view of the anterior end showing the5 anterior flagella (aF) arising as a bundle inside a gullet (arrow). Therecurrent flagellum is situated in a slit alongthe undulating membrane (UM)on thedorsalsideofthe body. Bar = 2 urn.- Fig. 5. Enlargementof theprominent UMwiththe recurrent flagellum (F) attached on the right side. The 5 anterior flagella arise at the anterior end. Bar = 5 urn. 0932-4739/9 4/0030-0372$3.50/0
© 1994 by Gustav Fischer Verlag, Stuttga rt
~
Ultrastructural study of Pentatrichomonoides . 373
374 . G. Brugerolle, A. Breunig and H. Konig Material and Methods The termites Mastotermes darwiniensis (Froggart) originating from Queensland in Australia wereobtained from the Bundesanstalt fur Materialforschung Berlin, Germany and they were cultured in glass vessels at 28° and fed on wood. Atermite wasdissected witha forceps andthe intestinal paunch opened in 1 mlof Ringer's solution to collect the intestinal fauna. The flagellates werepicked up with a glass pipette, observed by phase contrast microscopy and deposited on a slide for Giemsa staining or fixation for scanning (SEM) and transmission (TEM) electron microscopy. For SEM cells were fixed with 1 % (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 10 min, dehydrated in increasing concentrations of n-propanol (30 %, 50 %, 70 %, 90 %, 100% (v/v)) 10 min each and then critical point dried (Model FL9496, Balzers Union). In 10 steps dimethyloxypropane was removed by adding CO2 • The dried samples were sputter-coated with gold-palladium (technics Hummer V) and examined in a Zeiss microscope model 900 at 25 kV. For TEM, cells were fixed with 1 % glutaraldehyde in 0.1 M phosphate buffer, pH 7, for30 minat roomtemperature, washed twice in buffer and post-fixed with 1 % osmium tetroxide in 0.1 M phosphate buffer, pH 7, for 1 h. After washing in water, the fixed cells were separated into two pellets, one containing the small flagellates and the other the large ones, before preembedding in 1 % agar and staining "en bloc" with saturated uranyl acetate in ethanol 70 % for 30 min. Dehydration was continued in ethanolseries and propylene oxideprior to embedding in Epon 812. Ultrathin sections were cut with a Reichert OMU2 ultramicrotome and stained for 10 min with Reynold's leadcitratesolution. Photographs were takenwitha transmission electron microscope model 1200EX Jeol at 80 kV. The figures are presented as viewed from the tip of flagella and from the top and the outside of the cell, as recommended [2].
Results
1. Light and Scanning EM Cells of Pentatrichomonoides are recognizable among the diverse fauna of the flagellates of M. darwiniensis by the presence of an undulating membrane (UM) and by the presence of 5 anterior flagella. The cell body is generally slender or cigar-shaped with a truncated posterior end and a conspicuous UM spiralling around the cell body (Figs. la, 3). It displays changes of shape and can become massive with a UM running on the dorsal or convex side (Figs. lb, 2). The slender forms have a length of 30 urn long and a width of 5 urn and the length of the anterior flagella is about 20 urn. The massive forms are shorter but larger (20 urn) than the slender ones. The five anterior flagella beat synchronously like a bundle and the recurrent
flagellum associated with the undulating membrane generally has a free terminal part. The point where the anterior flagella emerge is surrounded by a groove (Figs. 2-4) opening to the dorsal side where the recurrent flagellum lies. This flagellum is intimately associated with the UM which follows a left-handed spiral UM (Figs. 3, 5). This UM is very prominent in this genus and a deep groove occurs on each side of it (Figs. 3, 5). There is no apparent axostyle or posterior axostylar spike like in other trichomonad cells. Giemsa staining (Fig. Ib) reveals the UM but does not stain the internal fibres or organelles other than the nucleus.
2. Electron microscopic observations The slender and the massive forms were also found in thin sections of fixed material (Figs. 6, 7). The slender forms clearly show the 5 free flagella arising at the anterior area and the recurrent flagellum adhering to the UM which reaches the posterior end. The striated costa follows the course of the UM and has a hook-like end (not shown). The cell membrane is underlain by a sheet of micro tubules except in the lamellar expansion forming the UM and on the two grooves situated on each side of the UM (Fig. 8). In the massive forms, the area free of microtubules seems to be more extensive and transformed in a pseudopodial zone where bacteria and food particles are endocytosed (Fig. 7). There is no axostylar tube or trunk distinguishable in cross sections (Fig. 8). The dictyosome is situated in the anterior half part of the body between the basal bodies and the nucleus (Figs. 6, 7). The cytoplasm is filled with digestive vacuoles, most containing bacteria or residual bodies, but no pieces of wood were detected (Figs. 6-8). The dense granules interpreted as hydrogenosomes are also recognizable (Fig. 8). Probably, some of the well-shaped and dividing bacteria are endosymbiotic (Fig. 6). Examination of sections of the anterior end shows that the 5 basal bodies of the free flagella are typically arranged around the basal body of the recurrent flagellum and can be numbered 1, 2, 3, 4, 5 following the trichomonad model (Figs. 9, 10). Basal body =#= 2 bears the preaxostylar or F2 fibres which distally subdivide into about 10 lamellae which are applied against the microtubules of the peltaaxostyle junction (Figs. 10, 11). These lamellae extend backwards and their lamellar shape differs from that usually found in other trichomonads. Also a typical fibrillar or X connection is seen between basal bodies =#= 1 and =#= 2 (Fig. 10). There are also two parallel rows of microtubules in the anterior area (Fig. 10). The inside one represents the pelta and develops forwards in the groove surrounding the anterior flagella (Figs. 7,9). The other
Fig. 6. Longitudinal section of a slender form showing the 5 free anteriorflagella, the recurrent flagellum (R), the dictyosome (G), the striatedcosta (C), the nucleus (N), the undulating membrane (UM) and several vacuoles, some containing bacteria (B). Bar = 1 lim. Fig. 7. Section of a massive form where the membrane isunderlain bya ribbonofaxostylarmicrotubules (Ax, enlarged in the leftupper edge) except on the UMsidewhere thecell forms numerous pseudopodia. Noticethe microtubules of thepelta-axostyle (PeAx) around the 5 anterior flagella in the gullet, the section of the costa (C) under the lamellar UM, the enlarged recurrent flagellum (R), the dictyosome (G), the nucleus (N) and the bacteria (B). Bar = 1 lim. - Fig. 8. Cross section of a slender form showing the row of microtubules (Ax) underlying the membrane except in the undulating membrane area (UM). Notice the presence of the costa (C), hydrogenosomes (H) and bacteria (B) in food vacuoles. Bar = 1 lim.
~
Ultrastructural study of Pentatrichomonoides . 375
UM
7
6
376 . G. Brugerolle, A. Breunig and H. Konig
makes up the microtubular row underlying the plasma membrane of most of the cell body surface and which is homologous to the axostyle. Several striated roots are attached to the basal bodies (Fig. 11). The largest one running under the UM corresponds to the costa. This fibre is transversely striated and displays thin dark bands alternating with large clear bands, the periodicity of the striation pattern being about 41 nm (Fig. 13a). By tilting the plane of the section a herring bone pattern (B pattern) or lattice appears in the large clear band (Fig. 13b). In transverse section the fibre seems to be composed of a stack of thin longitudinal lamellae and is surrounded by an amorphous layer (Fig. 14). In the posterior part, an amorphous internal zone enlarges progressively, replacing the striated portion of the fibre (Fig. 15). Several other striated fibres arise from the basal bodies (Fig. 11) one of which is situated under the surface microtubules and close to the cis-face of the dictyosome (Fig. 12) and could correspond to the "innominate fibre" described by Kirby in this genus. This fibre has the A pattern of striation typical of the parabasal fibres (not shown)though it is not in contact with the Golgi cisternae as in the parabasal apparatuses of other trichomonads. Only a very thin fibre makes close contact with the Golgi cisternae in this species (Fig. 12). The UM is typically composed of a lamellar expansion of the cell body associated with the recurrent flagellum and situated close to the costa . The lamella contains a dense structure or marginal lamella very similar to that occurring in the subfamily Trichomonadinae (Figs. 16, 17). The axoneme of the recurrent flagellum is associated with amorphous material attached to one side of the axoneme. A reconstruction diagram of the cell is presented in Fig. 18.
Discussion The trichomonad described here with 5 flagella, a conspicuous undulating membrane, and its characteristic cell body shape corresponds well to the genus Pentatrichomonoides described by Kirby [11] and Grasse [7] in different termite species. This strain occurring in Masto-
*'
termes has the same size as P. scroa described in Cryptotermes and could be the same species. Some features are more characteristic of Pentatrichomonoides, particularly the absence of a central axostyle
which seems to be represented by a microtubular row situated under the surface of the plasma-membrane. Microtubules arise from the anterior part which is called pelta-axostyle junction and are homologous to the microtubules of the hollow axostyle present in other genera although having a different shape and position [3,5, 10]. Due to the unusual position ofaxostylar microtubules a transverse section of Pentatrichomonoides is very similar to that of kinetoplastid flagellates such as Cryptobia [6] or Trypanosoma [16] which possess a corset of microtubules located under the plasma membrane. However, this character is a convergence, since the two lineages Parabasala and Kinetoplastida are well separated [18, 14]. Also the preaxostylar fibres which divides into lamellae are slightly different in shape from those of other trichomonads. Small differences in the ultrastructure and shape of these fibres have already been noted, they are cross striated in the Trichomonadinae [13], but not in the Tritrichomonadinae [3, 10], indicating a difference in their composition. This is corroborated by the absence of cross-reactivity of the antibodies which only decorate the preaxostylar fibres of the Tritrichomonas genus [18]. These specific ultrastructural characters and those regarding the cell shape justified the creation of a new genus by Kirby [11] and the new subfamily proposed later by Honigberg [9]. Examination of the ultrastructure of the costa with its so-called herring bone pattern and that of the lamellar shape of the UM shows that these characters are shared by the Trichomonadinae comprising the genera Trichomon-
as, Tetratrichomonas, Pentatrichomonas, Trichomitopsis and Pseudotrypanosoma [1,3,5,8,10]. The presence of a
dilated recurrent flagellum associated with a lamellar UM is only found in Trichomitopsis and Pseudotrypanosoma [1,8] which seem to be closely related to Pentatrichomonoides. All Trichomonadinae genera except Trichomitus form one branch of the main bifurcation demonstrated by rRNA sequence comparison in Trichomonadida, the other branch being represented by the Tritrichomonadinae and some associated genera such as Monocercomonas and
Hypotrichomonas [19].
Fig. 9. Section of the gulletcontainingthe 5 flagella, flagellum 2 adheresto a cytoplasmic expansion(arrow),the recurrentflagellum (R) ariseson the left side of the UM. Bar = 111m. - Fig. 10. The 5 basal bodiesof the anterior flagella (1,2,3,4,5) are orthogonally arranged around the basal body (R) of the recurrent flagellum. Basal body 2 bears the preaxostylar fibre (Pax) which subdividesin lamellae (arrow and upper left edge) located near the microtubules of the pelta-axostyle junction (rnt). A connection between basal bodies 1 and 2 is also present (arrowhead). Bar = 111m. - Fig. 11. The main striated fibre, the costa (C), is attached to the basal bodieslikeseveral other fibres (arrow). Thelamellaof the preaxostylarfibre (Pax) are seenalongthe axostylefar fromthe anterior end. Bar = 111m. - Fig. 12. This micrographshows the dictyosome in cross section (G) with the costa (C) on one side and the parabasal fibres (PF) on the other side; only the thin fibre of the parabasal body is in contact with the cis-face of the Golgi. Bar = 111m. Figs. 13a, 13b. Thesetwo photographsof the samelongitudinal sectionof the costadiffering in a 20°degrees tiltingof the section show the striation pattern of the fibre (13a) and the herring bone lattice in the clear band (arrow) when tilted (13b). Bar = 0.5 11m. Figs. 14, 15. A transverse sectionof the costa (Fig. 14) shows the lamellarstructure of the main part of the fibre (arrowhead) and the amorphouszoneon one sideand on the periphery. Alongitudinalsectionof the terminalpart (Fig. 15)shows the amorphouszonein the center of the fibre (arrow) and the periphery containing an additional striation (arrowhead) between the main dense bands. Bar = 0.2 11m and 111m, respectively. - Figs. 16, 17. Longitudinal and transverse sections of the lamellarundulatingmembrane (UM) containingthe densemarginallamellaand of the adherent recurrent flagellum where fibrillar materialis associated with the axoneme (arrow). Costa (C). Bar = 1 11m and 0.2 11m, respectively.
*'
*'
*'
~
Ultrastructural study of Pentatrichomonoides . 377
378 . G. Brugerolle, A. Breunig and H. Konig
Fig. 18. Diagram presentingthe cell organisation of Pentatricbomonoides. The 5 anterior free flagella (AF) arise at the anterior end from 5 kinetosomes (K). The recurrent flagellum (RF) is associated with a lamellar undulating membrane (UM) sustained by a striated fibre, the costa (C). The Golgi apparatus (G) is supported by parabasal fibers (PF) attached to the kinetosomes. Microtubules of the pelta encircle the gullet of the flagellar area and those forming the axostyle are situated under the plasma membrane around the cell except in the zone of the undulating membrane. The nucleus (N), the hydrogenosomes (H) and the food vacuoles (V) are represented.
The position of the fifth anterior flagellum in Pentatrichomonoides is similar to that of Hexamastix [3, 5], however, in the latter the UM and the costa are lacking and other features regarding the parabasal apparatus and the axostyle are different, which does not permi t the establishment of a direct evolutionary relationship between these genera.
References Amos W. B., Grimstone A. V., Rothschild L. J. and AllenR. D. (1979): Structure, protein composition and birefringence of the costa: a motile flagellar root fibre in the flagellate Trichomonas. J. Cell Sci., 35, 139-1 64. 2 Andersen R. A., Barr D. J. S., Lynn D. H., Melkonian M., Moestrup O. and Sleigh M. A. (1991): Terminology and nomenclature of the cytoskeletal elements associated with the flagellar/ciliary apparatu s in protists. Protoplasma, 164, 1-8 . 3 Brugerolle G. (1975): Cytologie ultrastructurale, systematique et evolution des Trichomonadida. Ann. Station de Besse en Chandesse, 10, 1-90. 4 Brugerolle G. (1986): Structural diversity of trichomonads as the basis for systematic and evolutionary considerations. Acta Universitatis Carolinae Biologica, 30, 199-210.
5 Brugerolle G. (1991): Flagellar and cytoskeletal systems in amitochondrial flagellates: Archamoebae, Metamonada and Parabasala. Protoplasma, 164, 70- 90. 6 Brugerolle G., Lorn J., Nohynkova E. et Joyon L. (1979): Comparaison et evolution des structures cellulaires chez plusieurs especes de Bodonides et Cryptobiides appartenant aux genres Bodo, Cryptobia et Trypanoplasma (Kinetoplastida, Mastigophora). Protistologica, 15, 197-221. 7 Grasse P. P. (1952): Ordre des Trichomonadines, phylogenie. In: Grasse P. P. (ed.): Traite de zoologie, vol. 1, pp. 704-779. Masson et Cie, Paris. 8 Hollande A. et Valentin J. (1968): Morphologic infrastructurale de Trichomonas (Trichomitopsis Kofoid et Swezy 1919) termopsidis, parasite intestinal de Termopsis angusticollis Walk. Critique de la notion de centrosome chez les Polymastigines. Protistologica, 4, 127-145. 9 Honigberg B. M. (1963): Evolutionary and systematic relationships in the flagellate order Trichomonadida Kirby. J. Protozool., 10, 20-63. 10 Honigberg B. M. and Brugerolle G. (1990): Structure. In: Honigberg B. M. (ed.): Trichomonads parasitic in humans, pp. 5-35 . Springer Verlag, New York. 11 Kirby H. (1931): Trichomonad flagellates from termites. II. Eutrichomastix, and the subfamily Trichomonadinae. Univ. California Pub. ZooL, 36, 171-262. 12 Lee J. J. (1988): Order Trichomonadida. In: LeeJ. J., Hutner S. H. and BoveeE. C. (eds.): Illustrated guide to the proto zoa, pp. 119-1 27. Society of Protozoologists, Lawrence, Kansas. 13 Nielsen M . H. (1972): Electron microscopy of Trichomonas vagina/is Donne. Negative staining of the mastigont. J. Microscopie, 15, 121-134. 14 Sogin M. (1991): Early evolution and origin of eukaryotes. Current Opinion in Genetics and Development, 1, 457-463. 15 Sutherland J. L. (1933): Protozoa from Australian termites. Quart . J. Mice. Sci., 76, 145-1 73. 16 Vickerman K. and Preston T. M. (1976): Comparative cell biology of the kinetoplastid flagellates . In: Lumsden W. H. R. and Evans D. A. (eds.): Biology of Kinetoplastida, vol. 1, pp. 35-130. Academic Press, London. 17 Viscogliosi E. and Brugerolle G. (1993a): Cytoskeleton in trichomonads. I. Immunological and biochemical comparative study of costal proteins in the genus Tritrichomonas. Europ. J. Protistol., 29, 160-1 70. 18 Viscogliosi E. and Brugerolle G. (1993b): Cytoskeleton in trichomonads. II. Immunological and biochemical characterization of the preaxostylar fibres and undulating membrane in the genus Tritrichomonas. Europ. J. Protistol., 29, 381-389. 19 Viscogliosi E., Philippe H., Baroin A., Perasso R. and Brugerolle G. (1993): Phylogeny of trichomonads based on partial sequences of large subunit rRNA and on cladistic analysis of morphological data. J. Euk. Microbiol., 40, 411-421. 20 Yamin M. (1979): Flagellates of the orders Trichomonadida Kirby, Oxymonadida Grasse, and Hypermastigida Grassi & Foa reported from lower termites (Isoptera families Mastotermitidae, Kalotermitidae, Hodot ermitidae, Termopsidae, Rhinotermitidae, and Serritermitidae) and from the woodfeeding roach Cryptocercus (Dictyoptera: Cryptocercidae). Sociobiology, 4, 3-11 7.
Key words: SEM - TEM - Cytoskeleton - Phylogenetic relationships Guy Brugerolle, Laboratoire de Biologic des Protistes, Universite Blaise Pascal de Clermont-Ferrand, 24 Av. des Landais, 63177 Aubiere Cedex, France
Ultrastructural Study of Pentatrichomonoides sp., a Trichomonad Flagellate from Mastotermes darwiniensis Guy Brugerolle1, Alfred Breunig 2 and Helmut K6nig 2 1 Laboratoire de Biologie des Protistes, Universite Blaise Pascal de Clermont-Ferrand, Aubiere, France 2 Angewandte Mikrobiologie, Universitat Ulm, VIm, Germany
SUMMARY The features of one trichomonad flagellate found in Mastotermes correspond to that of Pentatrichomonoides Kirby.The5 basal bodiesof the anterior flagella arearranged like in other trichomonads and particularly like in Hexamastix. The herring bone striation pattern of the costa and the lamellar shape of the UM are very closely related to those of the genus Trichomitopsis and, more generally said, to the Trichomonadinae genera: Trichomonas, Tetratrichomonas and Pentatrichomonas. Asignificant modificationconcernsthe microtubules ofthe axostylewhich do not forman axialhollow tube buta rowof microtubulesunderlying the plasma membrane except in the UM region which is the endocytotic area for the cell. Alsothe preaxostylar fibres subdivide into thin lamellae never seen in a trichomonad.
Introduction The parabasalid group represents a broad evolutionary lineage comprising the small trichomonads and the large hypermastigids. The extent and composition of the lineage are known from the studies of former authors using classical cytology and light microscopy [7, 9, 11, 12]. Many genera of trichomonads have been studied by modern techniques such as electron microscopy [3, 4, 8, 10] but those which compose the complex and varied fauna of the termites are difficult to collect and to screen and many genera have not been investigated. The completion of these comparative studies is necessary to establish the phylogeny of this group [19]. In addition it is very useful to study the biochemical and immunological diversification of the cytoskeleton in these flagellates [5, 17, 18].
We have had the opportunity to examine the fauna of the most primitive termite Mastotermes darwiniensis and to investigate a large trichomon ad corresponding to the genus Pentatrichomonoides. This genus and the species P. seroa were very precisely described by Kirby 1931 [11] in Cryptotermes dudley Banks and C. longieollis Banks of Panama and had been assigned to a new subfamily Penratrichomonoidinae by Honigberg in 1963 [9]. Though not reported by Sutherland 1933 [15] in Australian termites and by Yamin 1982 [20], a similar species has been recognized by Cleveland in Mastotermes darwiniensis (reported by Honigberg 1963 [9]) and a second one in Cryptotermes lamaniensis by Grasse 1952 [7]. Our study is in agreement to that of Kirby [11] and discloses some cytological peculiarities interesting for the compar ative cytology of trichomonad s and for their evolution [19].
Fig. 1. Light microscopicviews of Pentatrichomonoides sp.Phasecontrast photographofa slender cell(Fig. 1a)and Giemsastainingof a massive cell (Fig. 1b).The anterior flagella (af), theundulatingmembrane (urn) andthe nucleus (N) are recognizable. Bar = 10 !tm.Figs. 2, 3. Scanning EMviews of a massive form (Fig. 2) and a slender form (Fig. 3), which is spiralled and truncated at the posterior end. The 5 anterior free flagella and the undulating membrane (UM) are distinguishable. Bar = 5 urn. - Fig. 4. Enlarged view of the anterior end showing the5 anterior flagella (aF) arising as a bundle inside a gullet (arrow). Therecurrent flagellum is situated in a slit alongthe undulating membrane (UM)on thedorsalsideofthe body. Bar = 2 urn.- Fig. 5. Enlargementof theprominent UMwiththe recurrent flagellum (F) attached on the right side. The 5 anterior flagella arise at the anterior end. Bar = 5 urn. 0932-4739/9 4/0030-0372$3.50/0
© 1994 by Gustav Fischer Verlag, Stuttga rt
~
Ultrastructural study of Pentatrichomonoides . 373
374 . G. Brugerolle, A. Breunig and H. Konig Material and Methods The termites Mastotermes darwiniensis (Froggart) originating from Queensland in Australia wereobtained from the Bundesanstalt fur Materialforschung Berlin, Germany and they were cultured in glass vessels at 28° and fed on wood. Atermite wasdissected witha forceps andthe intestinal paunch opened in 1 mlof Ringer's solution to collect the intestinal fauna. The flagellates werepicked up with a glass pipette, observed by phase contrast microscopy and deposited on a slide for Giemsa staining or fixation for scanning (SEM) and transmission (TEM) electron microscopy. For SEM cells were fixed with 1 % (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 10 min, dehydrated in increasing concentrations of n-propanol (30 %, 50 %, 70 %, 90 %, 100% (v/v)) 10 min each and then critical point dried (Model FL9496, Balzers Union). In 10 steps dimethyloxypropane was removed by adding CO2 • The dried samples were sputter-coated with gold-palladium (technics Hummer V) and examined in a Zeiss microscope model 900 at 25 kV. For TEM, cells were fixed with 1 % glutaraldehyde in 0.1 M phosphate buffer, pH 7, for30 minat roomtemperature, washed twice in buffer and post-fixed with 1 % osmium tetroxide in 0.1 M phosphate buffer, pH 7, for 1 h. After washing in water, the fixed cells were separated into two pellets, one containing the small flagellates and the other the large ones, before preembedding in 1 % agar and staining "en bloc" with saturated uranyl acetate in ethanol 70 % for 30 min. Dehydration was continued in ethanolseries and propylene oxideprior to embedding in Epon 812. Ultrathin sections were cut with a Reichert OMU2 ultramicrotome and stained for 10 min with Reynold's leadcitratesolution. Photographs were takenwitha transmission electron microscope model 1200EX Jeol at 80 kV. The figures are presented as viewed from the tip of flagella and from the top and the outside of the cell, as recommended [2].
Results
1. Light and Scanning EM Cells of Pentatrichomonoides are recognizable among the diverse fauna of the flagellates of M. darwiniensis by the presence of an undulating membrane (UM) and by the presence of 5 anterior flagella. The cell body is generally slender or cigar-shaped with a truncated posterior end and a conspicuous UM spiralling around the cell body (Figs. la, 3). It displays changes of shape and can become massive with a UM running on the dorsal or convex side (Figs. lb, 2). The slender forms have a length of 30 urn long and a width of 5 urn and the length of the anterior flagella is about 20 urn. The massive forms are shorter but larger (20 urn) than the slender ones. The five anterior flagella beat synchronously like a bundle and the recurrent
flagellum associated with the undulating membrane generally has a free terminal part. The point where the anterior flagella emerge is surrounded by a groove (Figs. 2-4) opening to the dorsal side where the recurrent flagellum lies. This flagellum is intimately associated with the UM which follows a left-handed spiral UM (Figs. 3, 5). This UM is very prominent in this genus and a deep groove occurs on each side of it (Figs. 3, 5). There is no apparent axostyle or posterior axostylar spike like in other trichomonad cells. Giemsa staining (Fig. Ib) reveals the UM but does not stain the internal fibres or organelles other than the nucleus.
2. Electron microscopic observations The slender and the massive forms were also found in thin sections of fixed material (Figs. 6, 7). The slender forms clearly show the 5 free flagella arising at the anterior area and the recurrent flagellum adhering to the UM which reaches the posterior end. The striated costa follows the course of the UM and has a hook-like end (not shown). The cell membrane is underlain by a sheet of micro tubules except in the lamellar expansion forming the UM and on the two grooves situated on each side of the UM (Fig. 8). In the massive forms, the area free of microtubules seems to be more extensive and transformed in a pseudopodial zone where bacteria and food particles are endocytosed (Fig. 7). There is no axostylar tube or trunk distinguishable in cross sections (Fig. 8). The dictyosome is situated in the anterior half part of the body between the basal bodies and the nucleus (Figs. 6, 7). The cytoplasm is filled with digestive vacuoles, most containing bacteria or residual bodies, but no pieces of wood were detected (Figs. 6-8). The dense granules interpreted as hydrogenosomes are also recognizable (Fig. 8). Probably, some of the well-shaped and dividing bacteria are endosymbiotic (Fig. 6). Examination of sections of the anterior end shows that the 5 basal bodies of the free flagella are typically arranged around the basal body of the recurrent flagellum and can be numbered 1, 2, 3, 4, 5 following the trichomonad model (Figs. 9, 10). Basal body =#= 2 bears the preaxostylar or F2 fibres which distally subdivide into about 10 lamellae which are applied against the microtubules of the peltaaxostyle junction (Figs. 10, 11). These lamellae extend backwards and their lamellar shape differs from that usually found in other trichomonads. Also a typical fibrillar or X connection is seen between basal bodies =#= 1 and =#= 2 (Fig. 10). There are also two parallel rows of microtubules in the anterior area (Fig. 10). The inside one represents the pelta and develops forwards in the groove surrounding the anterior flagella (Figs. 7,9). The other
Fig. 6. Longitudinal section of a slender form showing the 5 free anteriorflagella, the recurrent flagellum (R), the dictyosome (G), the striatedcosta (C), the nucleus (N), the undulating membrane (UM) and several vacuoles, some containing bacteria (B). Bar = 1 lim. Fig. 7. Section of a massive form where the membrane isunderlain bya ribbonofaxostylarmicrotubules (Ax, enlarged in the leftupper edge) except on the UMsidewhere thecell forms numerous pseudopodia. Noticethe microtubules of thepelta-axostyle (PeAx) around the 5 anterior flagella in the gullet, the section of the costa (C) under the lamellar UM, the enlarged recurrent flagellum (R), the dictyosome (G), the nucleus (N) and the bacteria (B). Bar = 1 lim. - Fig. 8. Cross section of a slender form showing the row of microtubules (Ax) underlying the membrane except in the undulating membrane area (UM). Notice the presence of the costa (C), hydrogenosomes (H) and bacteria (B) in food vacuoles. Bar = 1 lim.
~
Ultrastructural study of Pentatrichomonoides . 375
UM
7
6
376 . G. Brugerolle, A. Breunig and H. Konig
makes up the microtubular row underlying the plasma membrane of most of the cell body surface and which is homologous to the axostyle. Several striated roots are attached to the basal bodies (Fig. 11). The largest one running under the UM corresponds to the costa. This fibre is transversely striated and displays thin dark bands alternating with large clear bands, the periodicity of the striation pattern being about 41 nm (Fig. 13a). By tilting the plane of the section a herring bone pattern (B pattern) or lattice appears in the large clear band (Fig. 13b). In transverse section the fibre seems to be composed of a stack of thin longitudinal lamellae and is surrounded by an amorphous layer (Fig. 14). In the posterior part, an amorphous internal zone enlarges progressively, replacing the striated portion of the fibre (Fig. 15). Several other striated fibres arise from the basal bodies (Fig. 11) one of which is situated under the surface microtubules and close to the cis-face of the dictyosome (Fig. 12) and could correspond to the "innominate fibre" described by Kirby in this genus. This fibre has the A pattern of striation typical of the parabasal fibres (not shown)though it is not in contact with the Golgi cisternae as in the parabasal apparatuses of other trichomonads. Only a very thin fibre makes close contact with the Golgi cisternae in this species (Fig. 12). The UM is typically composed of a lamellar expansion of the cell body associated with the recurrent flagellum and situated close to the costa . The lamella contains a dense structure or marginal lamella very similar to that occurring in the subfamily Trichomonadinae (Figs. 16, 17). The axoneme of the recurrent flagellum is associated with amorphous material attached to one side of the axoneme. A reconstruction diagram of the cell is presented in Fig. 18.
Discussion The trichomonad described here with 5 flagella, a conspicuous undulating membrane, and its characteristic cell body shape corresponds well to the genus Pentatrichomonoides described by Kirby [11] and Grasse [7] in different termite species. This strain occurring in Masto-
*'
termes has the same size as P. scroa described in Cryptotermes and could be the same species. Some features are more characteristic of Pentatrichomonoides, particularly the absence of a central axostyle
which seems to be represented by a microtubular row situated under the surface of the plasma-membrane. Microtubules arise from the anterior part which is called pelta-axostyle junction and are homologous to the microtubules of the hollow axostyle present in other genera although having a different shape and position [3,5, 10]. Due to the unusual position ofaxostylar microtubules a transverse section of Pentatrichomonoides is very similar to that of kinetoplastid flagellates such as Cryptobia [6] or Trypanosoma [16] which possess a corset of microtubules located under the plasma membrane. However, this character is a convergence, since the two lineages Parabasala and Kinetoplastida are well separated [18, 14]. Also the preaxostylar fibres which divides into lamellae are slightly different in shape from those of other trichomonads. Small differences in the ultrastructure and shape of these fibres have already been noted, they are cross striated in the Trichomonadinae [13], but not in the Tritrichomonadinae [3, 10], indicating a difference in their composition. This is corroborated by the absence of cross-reactivity of the antibodies which only decorate the preaxostylar fibres of the Tritrichomonas genus [18]. These specific ultrastructural characters and those regarding the cell shape justified the creation of a new genus by Kirby [11] and the new subfamily proposed later by Honigberg [9]. Examination of the ultrastructure of the costa with its so-called herring bone pattern and that of the lamellar shape of the UM shows that these characters are shared by the Trichomonadinae comprising the genera Trichomon-
as, Tetratrichomonas, Pentatrichomonas, Trichomitopsis and Pseudotrypanosoma [1,3,5,8,10]. The presence of a
dilated recurrent flagellum associated with a lamellar UM is only found in Trichomitopsis and Pseudotrypanosoma [1,8] which seem to be closely related to Pentatrichomonoides. All Trichomonadinae genera except Trichomitus form one branch of the main bifurcation demonstrated by rRNA sequence comparison in Trichomonadida, the other branch being represented by the Tritrichomonadinae and some associated genera such as Monocercomonas and
Hypotrichomonas [19].
Fig. 9. Section of the gulletcontainingthe 5 flagella, flagellum 2 adheresto a cytoplasmic expansion(arrow),the recurrentflagellum (R) ariseson the left side of the UM. Bar = 111m. - Fig. 10. The 5 basal bodiesof the anterior flagella (1,2,3,4,5) are orthogonally arranged around the basal body (R) of the recurrent flagellum. Basal body 2 bears the preaxostylar fibre (Pax) which subdividesin lamellae (arrow and upper left edge) located near the microtubules of the pelta-axostyle junction (rnt). A connection between basal bodies 1 and 2 is also present (arrowhead). Bar = 111m. - Fig. 11. The main striated fibre, the costa (C), is attached to the basal bodieslikeseveral other fibres (arrow). Thelamellaof the preaxostylarfibre (Pax) are seenalongthe axostylefar fromthe anterior end. Bar = 111m. - Fig. 12. This micrographshows the dictyosome in cross section (G) with the costa (C) on one side and the parabasal fibres (PF) on the other side; only the thin fibre of the parabasal body is in contact with the cis-face of the Golgi. Bar = 111m. Figs. 13a, 13b. Thesetwo photographsof the samelongitudinal sectionof the costadiffering in a 20°degrees tiltingof the section show the striation pattern of the fibre (13a) and the herring bone lattice in the clear band (arrow) when tilted (13b). Bar = 0.5 11m. Figs. 14, 15. A transverse sectionof the costa (Fig. 14) shows the lamellarstructure of the main part of the fibre (arrowhead) and the amorphouszoneon one sideand on the periphery. Alongitudinalsectionof the terminalpart (Fig. 15)shows the amorphouszonein the center of the fibre (arrow) and the periphery containing an additional striation (arrowhead) between the main dense bands. Bar = 0.2 11m and 111m, respectively. - Figs. 16, 17. Longitudinal and transverse sections of the lamellarundulatingmembrane (UM) containingthe densemarginallamellaand of the adherent recurrent flagellum where fibrillar materialis associated with the axoneme (arrow). Costa (C). Bar = 1 11m and 0.2 11m, respectively.
*'
*'
*'
~
Ultrastructural study of Pentatrichomonoides . 377
378 . G. Brugerolle, A. Breunig and H. Konig
Fig. 18. Diagram presentingthe cell organisation of Pentatricbomonoides. The 5 anterior free flagella (AF) arise at the anterior end from 5 kinetosomes (K). The recurrent flagellum (RF) is associated with a lamellar undulating membrane (UM) sustained by a striated fibre, the costa (C). The Golgi apparatus (G) is supported by parabasal fibers (PF) attached to the kinetosomes. Microtubules of the pelta encircle the gullet of the flagellar area and those forming the axostyle are situated under the plasma membrane around the cell except in the zone of the undulating membrane. The nucleus (N), the hydrogenosomes (H) and the food vacuoles (V) are represented.
The position of the fifth anterior flagellum in Pentatrichomonoides is similar to that of Hexamastix [3, 5], however, in the latter the UM and the costa are lacking and other features regarding the parabasal apparatus and the axostyle are different, which does not permi t the establishment of a direct evolutionary relationship between these genera.
References Amos W. B., Grimstone A. V., Rothschild L. J. and AllenR. D. (1979): Structure, protein composition and birefringence of the costa: a motile flagellar root fibre in the flagellate Trichomonas. J. Cell Sci., 35, 139-1 64. 2 Andersen R. A., Barr D. J. S., Lynn D. H., Melkonian M., Moestrup O. and Sleigh M. A. (1991): Terminology and nomenclature of the cytoskeletal elements associated with the flagellar/ciliary apparatu s in protists. Protoplasma, 164, 1-8 . 3 Brugerolle G. (1975): Cytologie ultrastructurale, systematique et evolution des Trichomonadida. Ann. Station de Besse en Chandesse, 10, 1-90. 4 Brugerolle G. (1986): Structural diversity of trichomonads as the basis for systematic and evolutionary considerations. Acta Universitatis Carolinae Biologica, 30, 199-210.
5 Brugerolle G. (1991): Flagellar and cytoskeletal systems in amitochondrial flagellates: Archamoebae, Metamonada and Parabasala. Protoplasma, 164, 70- 90. 6 Brugerolle G., Lorn J., Nohynkova E. et Joyon L. (1979): Comparaison et evolution des structures cellulaires chez plusieurs especes de Bodonides et Cryptobiides appartenant aux genres Bodo, Cryptobia et Trypanoplasma (Kinetoplastida, Mastigophora). Protistologica, 15, 197-221. 7 Grasse P. P. (1952): Ordre des Trichomonadines, phylogenie. In: Grasse P. P. (ed.): Traite de zoologie, vol. 1, pp. 704-779. Masson et Cie, Paris. 8 Hollande A. et Valentin J. (1968): Morphologic infrastructurale de Trichomonas (Trichomitopsis Kofoid et Swezy 1919) termopsidis, parasite intestinal de Termopsis angusticollis Walk. Critique de la notion de centrosome chez les Polymastigines. Protistologica, 4, 127-145. 9 Honigberg B. M. (1963): Evolutionary and systematic relationships in the flagellate order Trichomonadida Kirby. J. Protozool., 10, 20-63. 10 Honigberg B. M. and Brugerolle G. (1990): Structure. In: Honigberg B. M. (ed.): Trichomonads parasitic in humans, pp. 5-35 . Springer Verlag, New York. 11 Kirby H. (1931): Trichomonad flagellates from termites. II. Eutrichomastix, and the subfamily Trichomonadinae. Univ. California Pub. ZooL, 36, 171-262. 12 Lee J. J. (1988): Order Trichomonadida. In: LeeJ. J., Hutner S. H. and BoveeE. C. (eds.): Illustrated guide to the proto zoa, pp. 119-1 27. Society of Protozoologists, Lawrence, Kansas. 13 Nielsen M . H. (1972): Electron microscopy of Trichomonas vagina/is Donne. Negative staining of the mastigont. J. Microscopie, 15, 121-134. 14 Sogin M. (1991): Early evolution and origin of eukaryotes. Current Opinion in Genetics and Development, 1, 457-463. 15 Sutherland J. L. (1933): Protozoa from Australian termites. Quart . J. Mice. Sci., 76, 145-1 73. 16 Vickerman K. and Preston T. M. (1976): Comparative cell biology of the kinetoplastid flagellates . In: Lumsden W. H. R. and Evans D. A. (eds.): Biology of Kinetoplastida, vol. 1, pp. 35-130. Academic Press, London. 17 Viscogliosi E. and Brugerolle G. (1993a): Cytoskeleton in trichomonads. I. Immunological and biochemical comparative study of costal proteins in the genus Tritrichomonas. Europ. J. Protistol., 29, 160-1 70. 18 Viscogliosi E. and Brugerolle G. (1993b): Cytoskeleton in trichomonads. II. Immunological and biochemical characterization of the preaxostylar fibres and undulating membrane in the genus Tritrichomonas. Europ. J. Protistol., 29, 381-389. 19 Viscogliosi E., Philippe H., Baroin A., Perasso R. and Brugerolle G. (1993): Phylogeny of trichomonads based on partial sequences of large subunit rRNA and on cladistic analysis of morphological data. J. Euk. Microbiol., 40, 411-421. 20 Yamin M. (1979): Flagellates of the orders Trichomonadida Kirby, Oxymonadida Grasse, and Hypermastigida Grassi & Foa reported from lower termites (Isoptera families Mastotermitidae, Kalotermitidae, Hodot ermitidae, Termopsidae, Rhinotermitidae, and Serritermitidae) and from the woodfeeding roach Cryptocercus (Dictyoptera: Cryptocercidae). Sociobiology, 4, 3-11 7.
Key words: SEM - TEM - Cytoskeleton - Phylogenetic relationships Guy Brugerolle, Laboratoire de Biologic des Protistes, Universite Blaise Pascal de Clermont-Ferrand, 24 Av. des Landais, 63177 Aubiere Cedex, France