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Ultrastructure of a Colourless Flagellate, Phyllomitus apiculatusSkuja 1948 (Kinetoplastida)
Arch. Protistenkd. 132 (1986):
l~lO
VEB Gustav Fischer Verlag Jena
Institute of Biology of Inland 'Vaters, Academy of Sciences of the USSR, Borok
Ultrastructure of a Colourless Flagellate, Phyllomitus apiculatus SKUJA 1948 (Kinetoplastida) By A. P. MYLNIKOV With 18 Figures Key words: Phyllomitu8 apiculatu8" Electron microscopy; Cytopharynx; Mitochondrion; Mucocysts
Summary The ultrastructure of a colourless predatory flagellate Phyllomitu8 apiculatu8 was studied. Outwardly the cell is covered with a layer of compact glycocalyx. Under the cell membrane there is a system of pellicular microtubules linked by bridges. The flagellar kinetosomes give off two bands consisting of 3 and 5 microtubules, and one S-shaped band of 6~7 microtubules. The two heterodynamic flagella have paraxial rods and bear fine unbranched mastigonemes. The flagellar kinetosomes lie parallel to each other and are connected by amorphous material. Food ingestion occurs by a tubular cytopharynx. The flagellar pocket and the cytopharynx are covered with thin hairs similar to flagellar mastigonemes. The vesicular nucleus contains a complexely structured nucleolus. The nuclear chromatin is inconspicuous. The Golgi apparatus, endoplasmic reticulum, and contractile vacuole are of the usual structure. There is apparently a single branched mitochondrion with lamellar cristae. Kinetoplast-like parts of the mitochondrion are found in degraded individuals. In the cytoplasm, there are tubular mucocysts with amorphous contents and endoplasmic bacteria. The peculiarities of the ultrastructure of Ph. apiculatu8 confirm that this species belongs to the kinetoplastids.
Introduction At present, the ultrastructure of free-living protozoa, including the flagellates, is being intensively studied. Special attention is paid to representatives of the bodonids (suborder Bodonina HOLLANDE 1952). They are widely distributed in fresh and salt waters and soil, but less studied in comparison with the suborder Trypanosomatina KENT 1880. In a number of publications, the fine structure of some species of the genera Bodo, Pleuromonas, Spiromonas (BROOKER 1971; BURZELL 1975; KARPOV and ZHUKOV 1983; MACDoNALD and DARBYSHIRE 1977) and others has been analyzed in detail. Their belonging to the order Kinetoplastida HONIGBERG 1963, emend. VICKERMAN 1976 is not doubted (VICKERMAN 1976). The usual fresh water inhabitant Phyllomitus apiculatus is also of interest. Its ecology has been considered in a recent work of STEINBERG, LENHART and KLEE (1983). This organism is peculiar in that it is a clear-cut predator. Light microscope data show that Phyllomitu8 belongs to the bodonids, although a typical kinetoplast (as in Bodo) is not found in Phyllolnitus (MYLNIKOV 1983). The presence of a permanent cytostome and stinging organelles (ejectosomes, STEINBERG et al. 1983) is also not clear. 1
Arch. Protistenkd. Bd. 132
2
A. P. }IYLNIKOV
Material and Methods Phyllomitus was isolated from bottom waters of Rybinsk reservoir and cloned. Below are given the results of investigation of the strain P-4. The flagellates were cultivated in Petri dishes on PRATT medium. Suspension of bacteriovorous flagellates Parabodo nitrophillus was offered as food. The prey, in its turn, was cultivated in a medium containing Aerobacter aerogenes. The doubling time of the predator was 8-20 h at 20-22 °e. The maximum number of individuals was reached on the 5-8th d after inoculation. For the investigations, the prey and predator were separated in a layer of liquid by centrifugation. Living organisms were examined under a Peraval microscope (Zeiss) with interference optics. To detect the kinetoplast, the flagellates were fixed with SCHAUDINN'S reagent and stained after FEULGEN. For electron microscopic studies, a suspension of cells was collected and fixed with a mixture of 2 % OS04 and 0.6 % glutaraldehyde in 0.05 M cacodylate buffer for 15-30 min at 1°C. After dehydration in in a series of alcohols and water-free acetone, the cells were embedded into a mixture of Epon 812 and Araldite M. The sections were stained with uranyl acetate and lead citrate according to REYNOLDS and inspected with a JEM-IOO e microscope at 60 kV.
Results In culture, Phyllomitu8 shows a significant moving activity. The swimming trajectory in this flagellate is a zigzag. The onward movement of Phyllomitu8 is accompanied by body oscillations. After a contact with prey, Phyllomitu8 stops and ingests the prey as a whole in 5-20 s. The cytostome extends considerably during this act. At the very moment of being engulfed, the prey's flagella are still moving. Sometimes the predator's recurrent flagellum is seen to push the prey into the cytopharynx by spiral movements. After ingestion of several prey cells the predator undergoes binary longitudinal fission. If food is insufficient, Phyllomitu8 diminishes in body size and dies. The predatory mode of feeding of this organism is obligatory (MYLNIKOV 1983). Cannibalism has not been observed. No cysts have been found. The general morphology of the strain P-4 (with the exception of cytostome location) corresponds to the description of SKUJA (1948) and to the drawing of STEINBERG et al. (1983). The body length is 8-15 pm, width 3-6 pm. The cells are oval or ovalelongate, with a large outgrowth of the body (the lip). The location of the main organelles is shown in Figs. 1, 2 and 8. Ourwardly the cell is covered with a layer of dense glycocalyx, 30 nm thick (Fig. 9). This layer is absent from only a small region of the flagellar pocket. Under the plasmalemma there is a system of pellicular microtubules linked by bridges (Figs. 9 and 11). The distances between the microtubule centers are equal 50-60 nm. Most of these microtubules have longitudinal orientation. The two heterodynamic flagella emerge from a depression, the flagellar pocket (Figs. 2, 5, 6 and 8). During swimming one flagellum (9-11pm long) is usually directed forward and performs rowing movements. The second flagellum (12-16 pm) is directed backwards; at the moment of movement cessation, it spirally winds around the flagellate's body. The flagella become thinner at their distal ends (Fig. 3). Thin mastigonemes are observed along the entire length of the flagella (Fig. 4). The mastigonemes are 200-250 nm long and 4.0-4.5 nm thick. The flagellar kinetosomes are oriented parallel to each other (Figs. 5 and 6). Cross-striated filaments have not been found. The kinetosomes are interconnected by an amorphous osmiophilic material. The flagellar basal plate rises over the cell surface by 190-230 nm. The flagellar surface is covered with the same layer of glycocalyx as the entire cell. Both flagella possess paraxial rods (Figs. 6 and 7) emerging at the level of the basal plate.
3
Ultrastructure of Phyllomitu8 apiculatu8 SKUJA 1948
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Fig. 1. Scheme of structure of Phyllomitu8 apiculatu8. A - general view, front end is-partly cut off; B - cross section of anterior body part; C - cross section on the nucleus level; D - cross section of posterior body end. Designations: f - flagella; th - thin hairs; co - cytopharynx opening; bm - S-shaped band of microtubules; cyt - cytopharynx; me - mucocyst; s - spur; psm - pellicular system of microtubules; n - nucleus; fv - food vacuole; Ga - Golgi apparatus; cv - contractile vacuole; mp - microtubular prism; m -- mitochondrion.
Several bands of microtubules starting near the kinetosomes have been found in the cell. Two bands, consisting of 3-5 microtubules each, run from the kinetosomes to the rear of the body. The third S-shaped band, consisting of 6-7 microtubules, underlines the lateral wall of the flagellar pocket and runs in a groove (Figs. 5, 8 and 11). At the anterior end of the body it bends backwards and passes under the cytopharyngeal membrane, accompanying the cytopharynx along its entire course (Figs. 11 and 13). At the bending point this band forms a small bulge on the body surface, which carries a bundle of thin sinuous filaments (Fig. 12). Near the S-shaped band of microtubules, a microtubular prism (terminology of BURZELL 1975) consisting of three rows of 6, 8 and 9 microtubules, and an osmiophilic amorphous structure 1*
4
A. P.
l\1YLNIKOV
(the spur) have been detected (Figs. 5, 9, 11 and 12). The spur accompanies the cytopharynx along its entire course (Figs. 5, 11 and 12). The cytopharynx starts at the anterior end as a small aperture and passes as a narrow tube parallel to the flagellar pocket (Figs. 2, 8, 10 and 11). Its maximum length is 4,um. The cytopharyngeal surface carries thin threads similar to flagellar mastigonemes. The narrow end of the cytopharynx is detected at the nucleus level. Since profiles of the flagellar pocket and cytopharynx are frequently observed on sections simultaneously, it is possible to draw a conclusion that these two depressions are not connected with each other. The Golgi apparatus (dimensions 0.3-0.5 X 1.0-1.2,um) and the endoplasmic reticulum are of the usual structure. The contractile vacuole is located between the cytopharynx and flagellar pocket (Fig. 8). During diastole stage, the contractile vacuole and the flagellar pocket are separated with only a thin partition. The contractile vacuole probably discharges its contents into the flagellar pocket. Food vacuoles lie most frequently in the posterior part of the body. Remnants of prey or bacteria have
Fig. 2. Longitudinal body section of Phyllomitus apiculatus. Designations: cyt - cytopharynx; cv - contractile vacuole; f - flagella; fp - flagellar pocket; fv - food vacuole; Ga - Golgi apparatus; m - mitochondrion; bm- band of microtubules; mc - mucocyst; n - nucleus; sbsymbiotic bacterium. Fig. 3. Dried preparation shows cytopharynx opening (co) and flagellar acronemes (fa). Fig. 4. Dried preparation shows mastigonemes and adhering bacterium (b). Fig. 5. Flagellar kinetosomes give off the spur (s) and the S-shaped band of microtubules (bm) passing in a groove. Band of 5 microtubules is seen (arrow). Flagellar kinetosomes (k) lie parallel to each other. Fig. 6. Flagellar kinetosomes lie parallel to each other. One kinetosome gives off the spur (s). Fig. 7. Flagella and flagellar pocket are covered with a layer of dense glycocalyx (dg). Paraxial rods are turned to each other. 'The wall of the flagellar pocket is strengthened by two bands of microtubules (bm). Figs. 8 and 9. Band of microtubules (bm) and microtubular prism (mp) run along the cytopharynx (cyt) and flagellar pocket (fp). Contractile vacuole (cv) and Golgi apparatus (Ga) lie between cytopharynx and flagellar pocket. lVIucocysts (me) are arranged in two rows. Pellicular microtubules (pm) are linked by bridges. Cell surface is covered with a layer of dense glycocalyx (dg). Fig. 10. 'The section shows elongated cytopharynx (cyt). Fig. II. Microtubular prism (mp), spur (s) and band of microtubules (bm) pass along the cytopharynx. A symbiotic bacterium (sb) and system of pellicular microtubules (psm) are seen. Fig. 12. 'The spur (s) lies along the cytopharynx. The cytopharynx opening is surrounded with thin hairs (th). Fig. 13. Band of microtubules (bm) passes along the wall of the flagellar pocket (fp), thcn it bends (arrow) and continues along the cytopharynx (cyt). Fig. 14. In a large mitochondrion (m) there are osmiophilic bodies (ob); lamellar cristae (c) are scattered in the peripheral part of the organelle. The envelope of the nucleus (n) shows nuclear pores. Fig. 15. The section shows the mitochondrion (m) filled with fibrous material (fm). Fig. 16. Longitudinal section through mucocysts (mc). Nearby, a profile of the mitochondrion (m). Fig. 17. The Golgi apparatus (Ga) secretes vesicles (v) which give rise to mucocysts. Fig. 18. Extruded mucocysts have reticulate walls.
Ultrastructure of Phyllomitu8 apiculatu8
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Ultrastructure of Phyllomitu8 apiculatu8
SKUJA
1948
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A. P.
MYLNIKOV
not been observed inside them. This can be explained by the fact that Phyllomitus was fixed at stationary growth phase and was not feeding. It has been impossible to observe the moment of food ingestion on sections. The nucleus, 1.8-2.2 pm in diameter, is situated near the flagellar kinetosomes. The nucleolus consists of several lobes, and the peripheral lobes surround the central part of the nucleolus (Figs. 2 and 14). The chromatin is inconspicuous. It is possible, however, the peripheral lobes consist of the chromatin. The nuclear pores can be noticed on several sections (Fig. 14). No stages of mitosis have been observed. The whole cell of the flagellate is permeated by a mitochondrion with lamellar cristae. The maximum length of the mitochondrion is 5-6 pm, the width being 2 pm. Besides the cristae, osmiophilic bodies and matrix are observed in the mitochondrion (Figs. 14-16). A typical kinetoplast has not been found. However, parts of the mitochondrion with condensed fibrous material resembling DNA have been noted on a number of sections (Fig. 15). Usually such kinetoplast-like structures are found in degraded or dead individuals. The diameter of this structure is equal to 1.5-2.0 pm. It has not been possible to detect mitochondrial DNA using FEULGEN'S method. Usually one mitochondrial profile is seen on sections of the anterior body end, and several profiles at the rear end. It is possible that the mitochondrion is branching near the rear end. At the anterior end there are peculiar trichocyst-like structures, here called mucocysts (according to HovAssE and MIGNOT 1975). The mucocysts are usually arranged in rows near the cytopharynx and in the lip (Figs. 2 and 8). There are 7-11 mucocysts in one row. Under the light microscope the mucocysts are seen after staining with haematoxylin or when using interference optics. A mucocyst consists of an outer tube, 120-150 nm in diameter and 2.5 pm long, and an inner amorphous tube 100 mn in diameter. A thread is seen inside the mucocyst. Between the tubes there is a bright zone (Figs. 16 and 17). It has not been possible to observe the process of liberation of the mucocysts. However, numerous extruded mucocysts have been fixed near the cells. They consist of a reticulate tube without inner contents (Fig. 18). The mesh size of the reticulum is 25 nm. The diameter of the reticulate tube is 150-200 nm. Thus, the diameter of the mucocysts increases after the liberation. The mucocysts are formed in the cisternae of the Golgi apparatus (Fig. 17). Extruded mucocysts are often seen inside the food vacuoles. Within the cells there are also symbiotic bacteria, 450 nm long and 300 nm in diameter (Fig. 11). It has been possible to observe division of these bacteria in the cytoplasm.
Discussion The following distinguishing features of the fine structure of Phyllomitus have been revealed in this work: paraxial rods in the flagella, subpellicular system of microtubules, three bands of kinetosome-associated microtubules, giant mitochondrion with lamellar cristae, tubular cytopharynx. All these features bring our species closer to the free-living bodonids whose ultrastructure has already been studied. This is primarily the case with Bodo caudatus, B. saltans (BROOKER 1971), B. curvifilus (BURZELL 1975), B. designis (EYDEN 1977), Pleuromonas jaculans (ANIKUSHIN 1975; KARPOV and ZHUKOV 1983) and also Rhynchomonas nasuta (SWALE 1973). Our investigations have revealed in Phyllomitus a layer of glycocalyx found in trypanosomes at certain stages of the life cycle (VICKERMAN and PRESTON 1976). This layer covers the flagella as well. It is believed to protect the trypanosomes from the host's antibodies. Its function in our predator is not clear.
Ultrastructure of Phyllomitu8 apiculatu8
SKUJA
1948
9
The free-living bodonids are characterized by the presence of a permanent cytopharynx which has the shape of a tube. Besides, it is covered with thin hairs similar to flagellar mastigonemes (KARPOV and ZHUKOV 1983). The same is observed in Phyllomitus. Like in the bodonids, the cytopharyngeal wall is reinforced in Phyllomitus by microtubules joined into bands consisting of 3, 5, and 6-7 microtubules. The last band is S-shaped. Practically the same kind of bands is known in the bodonids and cryptobiids (BRUGEROLLE et al. 1979; KARPOV and ZHUKOV 1983). The microtubular prism in Phyllomitus is similar to that in Bodo curvifilus and B. designis (BURZELL 1975; EVDEN 1977). The spur emerging from the kinetosome in Phyllomitus is also found in the euglenoid alga Isonema nigricans (SCHUSTER et al. 1968). EVDEN (1977) thinks that the microtubular prism is probably homologous to the band of 20 microtubules in Bodo spp. (BROOKER 1971) and to the quadritubular band in Cephalothamnium cyclopum (HITCHEN 1974). The flagellar pocket is the second depression on the Phyllomitus body. The heterodynamic flagella emerge from the bottom of the pocket. Such an arrangement of the flagella, i.e. parallel position of the kinetosomes, as well as the fact that the contractile vacuole discharges its contents into the flagellar pocket are characteristic of the kinetoplastids. The mastigonemes of Phyllomitus are similar to those in Pleuromonas jaculans (KARPOV and ZHUKOV 1983) but are less conspicuous than the mastigonemes in Bodo saltans and B. caudatus (BROOKER 1971). The nucleus, Golgi apparatus, contractile vacuole, and endoplasmic reticulum in Phyllomitus are typical of the kinetoplastids. One of the interesting features in Phyllomitus is the absence of a typical kinetoplast. Kinetoplast-like structures are found rarely and only in a few individuals. Probably such an organisation of the mitochondrion is more like that in the bodonid Ichthyobodo necator (JOVON and LOM 1969). Osmiophilic bodies in the mitochondrion of Phyllomitus are known to occur in the bodonid Spiromonas angusta (MACDoNALD and DARBVSHIRE 1977). The latter has other elements of morphology similar to those in Phyllomitus: heterodynamic flagella, cytopharynx with underlining microtubules, subpellicular microtubules, and a bundle of filaments near the cytopharynx opening. Moreover, the cytopharynx opening in S. angusta is situated at the anterior end of the body, like in Phyllomitus. Recently STEINBERG et al. (1983) noted stinging organelles at the anterior end of the body in Phyllomitus. Our electron micrographs show the structure of these organelles. Similar organelles are known in many Protozoa: ciliates, dinoflagellates, chloromonads, euglenomonadids (HAUSMANN 1978), but not in the bodonids. An especially great similarity in size and shape is observed between the mucocysts of Phyllomitus and those in euglenoid algae Isonema nigricans (SCHUSTER et al. 1968) and Entosiphon sulcatum (MIGNOT and HOVASSE 1973). The tubular wall of these organelles has a reticulate structure. They are filled with amorphous material. Somewhat larger mucocysts with a reticulate wall are found in Peranema trichophorum (HILENSKI and WALNE 1983). The function of the mucocysts in Phyllomitus is revealed still incompletely. The moment of extrusion of mucocysts has not been fixed by us. No prey stricken by a mucocysts has been observed. However, the presence of empty mucocysts in food vacuoles and the location of mature mucocysts close to the cytopharynx probably indicate participation of these organelles in food ingestion. Thus, our data have shown that the structure plan and the peculiarities of the ultrastructure of Phyllomitus allow to come to a conclusion that this species belongs to the suborder Bodonina of the order Kinetoplastida.
10
A. P. MYLNIKOV, Ultrastructure of Phyllomitu8 apiculatu8 SK1:"JA 1948
Literature ANIKUSHIN, N. F. (1975): Peculiarities of the ultrastructure of the mitochondrion in flagellates of the suborder Bodonina. Cytology 17: 94-95 (in Russian). BRUGEROLLE, G., LOM, J., NOHYNKOVA, E., et JOYON, L. (1979): Comparaison et evolution des structures cellulaires ches plusieurs especes de bodonides et cryptobiides appartenant aux genres Bodo, Cryptobia et Trypanopla8ma (Kinetoplastida, Mastigophora). Protistologica 15: 197-221. BROOKER, B. E. (1971): Fine structure of Bodo 8altan8 and Bodo caudatu8 (Zoomastigophorea: Protozoa) and their affinities with the Trypanosomatidae. Bull. Brit. Mus. (Natur. Hist.) Zoo!' 22: 87-102. BURZELL, Z. A. (1975): Fine structure of Bodo curvifilu8 GRIESS MANN (Kinetoplastida: Bodonidae). J. Protozoo!. 22: 35-39. EYDEN, B. P. (1977): Morphology and ultrastructure of Bodo de8igni8 SKUJA, 1948. Protistologica 13: 169-179. HAUSMANN, K. (1978): Extrusive organelles in protists. Int. Rev. Cyto!. 52: 197-276. HILENSKI, L. L., and W ALNE, P. L. (1983): Ultrastructure of mucocysts in Peranema trichophorum (Euglenophyceae). J. Protozoo!. 30: 491-496. HITCHEN, E. T. (1974): The fine structure of the colonial kinetoplastid flagellate Cephalothamnium cyclopum STEIN. J. Protozoo!. 21: 221-231. HOVASSE, R., et MIGNOT, J. P. (1975): Trichocystes et organites analogues ches les Protistes. Ann. Bio!. 14: 397-422. JOYON, L., et LOM, L. (1969): Etude cytologique, systematique et pathologique d'Ichthyobodo necator (HENNEGU¥, 1883) PINTO, 1928 (zooflagelle). J. Protozoo!. 16: 703-719. KARPOV, S. A., and ZHUKOV, B. F. (1983): The ultrathin structure of Pleuromona8 jaculan8 PERTY (Kinetoplastida, Zoomastigophorea, Sarcomastigophora). In: N. N. BANINA, T. V. BEYER and K. M. SUKHANOVA (eds.), Protozoa of activated sludge, Leningrad: 153-156 (in Russian). MAcDoNALD, C. M., and DARBYSHIRE, J. F. (1977): The morphology of a soil flagellate, Spiromona8 angu8ta (DuJ.) ALEXEIEFF (Mastigophorea: Protozoa). Protistologica 13: 441-450. MIGNOT, J. P., et HOVASSE, R. (1973): Nouvelle contribution a la connaissance des trichocystes: les organes grillages d'Ento8iphon 8ulcatum (Flagellata, Euglenida). Protistologica 9: 371-39l. :VIYLNIKOV, A. P. (1983): Feeding of predatory zooflagellates. BioI. vnutr. vod, Inform. bull. No. 60: 33-37 (in Russian). SCHUSTER, F. L., GOLDSTEIN, S., and HERSHENOV, B. (1968): Ultrastructure of a flagellate, 180nema nigrican8 nov. gen. nov. sp., from a polluted marine habitat. Protistologica 4: 141-149. SKUJA, N. (1948): Taxonomic des Phytoplanktons einiger Seen in Uppland, Schweden. Symb. Bot. Upsa!. 9/3: 1-399. STEINBERG, CH., LENHART, B., und KLEE, R. (1983): Bemerkungen zur Okologie cines farblosen Phytoflagellaten, Phyllomitu8 apiculatu8 SKUJA (1948), Cryptophyceae. Arch. Protistenkd. 127: 307-317. SWALE, E. 1\1. F. (1973): A study of the colourless flagellate Rhynchomona8 na8uta (STOKES) KLEBS. Bio!. J. Linn. Soc. 5: 255-264. VICKERJ\IAN, K. (1976): The diversity of the kinetoplastid flagellates. In: W. H. R. LUMSDEN and D. A. EVANS (cds.), Biology of kinetoplastida, pp. 1-34. London, New York, San Francisco. and PRESTON, T. 1\T. (1976): Comparative cell biology of kinetoplastid flagellates. Ibid. pp. 35-130. Author's address: A. P. MYLNIKOV, Laboratory of Biology of Lower Organisms, Institute of Biology of Inland Waters, USSR Academy of Sciences, 152742 Borok, Jaroslavl District, USSR.
l~lO
VEB Gustav Fischer Verlag Jena
Institute of Biology of Inland 'Vaters, Academy of Sciences of the USSR, Borok
Ultrastructure of a Colourless Flagellate, Phyllomitus apiculatus SKUJA 1948 (Kinetoplastida) By A. P. MYLNIKOV With 18 Figures Key words: Phyllomitu8 apiculatu8" Electron microscopy; Cytopharynx; Mitochondrion; Mucocysts
Summary The ultrastructure of a colourless predatory flagellate Phyllomitu8 apiculatu8 was studied. Outwardly the cell is covered with a layer of compact glycocalyx. Under the cell membrane there is a system of pellicular microtubules linked by bridges. The flagellar kinetosomes give off two bands consisting of 3 and 5 microtubules, and one S-shaped band of 6~7 microtubules. The two heterodynamic flagella have paraxial rods and bear fine unbranched mastigonemes. The flagellar kinetosomes lie parallel to each other and are connected by amorphous material. Food ingestion occurs by a tubular cytopharynx. The flagellar pocket and the cytopharynx are covered with thin hairs similar to flagellar mastigonemes. The vesicular nucleus contains a complexely structured nucleolus. The nuclear chromatin is inconspicuous. The Golgi apparatus, endoplasmic reticulum, and contractile vacuole are of the usual structure. There is apparently a single branched mitochondrion with lamellar cristae. Kinetoplast-like parts of the mitochondrion are found in degraded individuals. In the cytoplasm, there are tubular mucocysts with amorphous contents and endoplasmic bacteria. The peculiarities of the ultrastructure of Ph. apiculatu8 confirm that this species belongs to the kinetoplastids.
Introduction At present, the ultrastructure of free-living protozoa, including the flagellates, is being intensively studied. Special attention is paid to representatives of the bodonids (suborder Bodonina HOLLANDE 1952). They are widely distributed in fresh and salt waters and soil, but less studied in comparison with the suborder Trypanosomatina KENT 1880. In a number of publications, the fine structure of some species of the genera Bodo, Pleuromonas, Spiromonas (BROOKER 1971; BURZELL 1975; KARPOV and ZHUKOV 1983; MACDoNALD and DARBYSHIRE 1977) and others has been analyzed in detail. Their belonging to the order Kinetoplastida HONIGBERG 1963, emend. VICKERMAN 1976 is not doubted (VICKERMAN 1976). The usual fresh water inhabitant Phyllomitus apiculatus is also of interest. Its ecology has been considered in a recent work of STEINBERG, LENHART and KLEE (1983). This organism is peculiar in that it is a clear-cut predator. Light microscope data show that Phyllomitu8 belongs to the bodonids, although a typical kinetoplast (as in Bodo) is not found in Phyllolnitus (MYLNIKOV 1983). The presence of a permanent cytostome and stinging organelles (ejectosomes, STEINBERG et al. 1983) is also not clear. 1
Arch. Protistenkd. Bd. 132
2
A. P. }IYLNIKOV
Material and Methods Phyllomitus was isolated from bottom waters of Rybinsk reservoir and cloned. Below are given the results of investigation of the strain P-4. The flagellates were cultivated in Petri dishes on PRATT medium. Suspension of bacteriovorous flagellates Parabodo nitrophillus was offered as food. The prey, in its turn, was cultivated in a medium containing Aerobacter aerogenes. The doubling time of the predator was 8-20 h at 20-22 °e. The maximum number of individuals was reached on the 5-8th d after inoculation. For the investigations, the prey and predator were separated in a layer of liquid by centrifugation. Living organisms were examined under a Peraval microscope (Zeiss) with interference optics. To detect the kinetoplast, the flagellates were fixed with SCHAUDINN'S reagent and stained after FEULGEN. For electron microscopic studies, a suspension of cells was collected and fixed with a mixture of 2 % OS04 and 0.6 % glutaraldehyde in 0.05 M cacodylate buffer for 15-30 min at 1°C. After dehydration in in a series of alcohols and water-free acetone, the cells were embedded into a mixture of Epon 812 and Araldite M. The sections were stained with uranyl acetate and lead citrate according to REYNOLDS and inspected with a JEM-IOO e microscope at 60 kV.
Results In culture, Phyllomitu8 shows a significant moving activity. The swimming trajectory in this flagellate is a zigzag. The onward movement of Phyllomitu8 is accompanied by body oscillations. After a contact with prey, Phyllomitu8 stops and ingests the prey as a whole in 5-20 s. The cytostome extends considerably during this act. At the very moment of being engulfed, the prey's flagella are still moving. Sometimes the predator's recurrent flagellum is seen to push the prey into the cytopharynx by spiral movements. After ingestion of several prey cells the predator undergoes binary longitudinal fission. If food is insufficient, Phyllomitu8 diminishes in body size and dies. The predatory mode of feeding of this organism is obligatory (MYLNIKOV 1983). Cannibalism has not been observed. No cysts have been found. The general morphology of the strain P-4 (with the exception of cytostome location) corresponds to the description of SKUJA (1948) and to the drawing of STEINBERG et al. (1983). The body length is 8-15 pm, width 3-6 pm. The cells are oval or ovalelongate, with a large outgrowth of the body (the lip). The location of the main organelles is shown in Figs. 1, 2 and 8. Ourwardly the cell is covered with a layer of dense glycocalyx, 30 nm thick (Fig. 9). This layer is absent from only a small region of the flagellar pocket. Under the plasmalemma there is a system of pellicular microtubules linked by bridges (Figs. 9 and 11). The distances between the microtubule centers are equal 50-60 nm. Most of these microtubules have longitudinal orientation. The two heterodynamic flagella emerge from a depression, the flagellar pocket (Figs. 2, 5, 6 and 8). During swimming one flagellum (9-11pm long) is usually directed forward and performs rowing movements. The second flagellum (12-16 pm) is directed backwards; at the moment of movement cessation, it spirally winds around the flagellate's body. The flagella become thinner at their distal ends (Fig. 3). Thin mastigonemes are observed along the entire length of the flagella (Fig. 4). The mastigonemes are 200-250 nm long and 4.0-4.5 nm thick. The flagellar kinetosomes are oriented parallel to each other (Figs. 5 and 6). Cross-striated filaments have not been found. The kinetosomes are interconnected by an amorphous osmiophilic material. The flagellar basal plate rises over the cell surface by 190-230 nm. The flagellar surface is covered with the same layer of glycocalyx as the entire cell. Both flagella possess paraxial rods (Figs. 6 and 7) emerging at the level of the basal plate.
3
Ultrastructure of Phyllomitu8 apiculatu8 SKUJA 1948
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Fig. 1. Scheme of structure of Phyllomitu8 apiculatu8. A - general view, front end is-partly cut off; B - cross section of anterior body part; C - cross section on the nucleus level; D - cross section of posterior body end. Designations: f - flagella; th - thin hairs; co - cytopharynx opening; bm - S-shaped band of microtubules; cyt - cytopharynx; me - mucocyst; s - spur; psm - pellicular system of microtubules; n - nucleus; fv - food vacuole; Ga - Golgi apparatus; cv - contractile vacuole; mp - microtubular prism; m -- mitochondrion.
Several bands of microtubules starting near the kinetosomes have been found in the cell. Two bands, consisting of 3-5 microtubules each, run from the kinetosomes to the rear of the body. The third S-shaped band, consisting of 6-7 microtubules, underlines the lateral wall of the flagellar pocket and runs in a groove (Figs. 5, 8 and 11). At the anterior end of the body it bends backwards and passes under the cytopharyngeal membrane, accompanying the cytopharynx along its entire course (Figs. 11 and 13). At the bending point this band forms a small bulge on the body surface, which carries a bundle of thin sinuous filaments (Fig. 12). Near the S-shaped band of microtubules, a microtubular prism (terminology of BURZELL 1975) consisting of three rows of 6, 8 and 9 microtubules, and an osmiophilic amorphous structure 1*
4
A. P.
l\1YLNIKOV
(the spur) have been detected (Figs. 5, 9, 11 and 12). The spur accompanies the cytopharynx along its entire course (Figs. 5, 11 and 12). The cytopharynx starts at the anterior end as a small aperture and passes as a narrow tube parallel to the flagellar pocket (Figs. 2, 8, 10 and 11). Its maximum length is 4,um. The cytopharyngeal surface carries thin threads similar to flagellar mastigonemes. The narrow end of the cytopharynx is detected at the nucleus level. Since profiles of the flagellar pocket and cytopharynx are frequently observed on sections simultaneously, it is possible to draw a conclusion that these two depressions are not connected with each other. The Golgi apparatus (dimensions 0.3-0.5 X 1.0-1.2,um) and the endoplasmic reticulum are of the usual structure. The contractile vacuole is located between the cytopharynx and flagellar pocket (Fig. 8). During diastole stage, the contractile vacuole and the flagellar pocket are separated with only a thin partition. The contractile vacuole probably discharges its contents into the flagellar pocket. Food vacuoles lie most frequently in the posterior part of the body. Remnants of prey or bacteria have
Fig. 2. Longitudinal body section of Phyllomitus apiculatus. Designations: cyt - cytopharynx; cv - contractile vacuole; f - flagella; fp - flagellar pocket; fv - food vacuole; Ga - Golgi apparatus; m - mitochondrion; bm- band of microtubules; mc - mucocyst; n - nucleus; sbsymbiotic bacterium. Fig. 3. Dried preparation shows cytopharynx opening (co) and flagellar acronemes (fa). Fig. 4. Dried preparation shows mastigonemes and adhering bacterium (b). Fig. 5. Flagellar kinetosomes give off the spur (s) and the S-shaped band of microtubules (bm) passing in a groove. Band of 5 microtubules is seen (arrow). Flagellar kinetosomes (k) lie parallel to each other. Fig. 6. Flagellar kinetosomes lie parallel to each other. One kinetosome gives off the spur (s). Fig. 7. Flagella and flagellar pocket are covered with a layer of dense glycocalyx (dg). Paraxial rods are turned to each other. 'The wall of the flagellar pocket is strengthened by two bands of microtubules (bm). Figs. 8 and 9. Band of microtubules (bm) and microtubular prism (mp) run along the cytopharynx (cyt) and flagellar pocket (fp). Contractile vacuole (cv) and Golgi apparatus (Ga) lie between cytopharynx and flagellar pocket. lVIucocysts (me) are arranged in two rows. Pellicular microtubules (pm) are linked by bridges. Cell surface is covered with a layer of dense glycocalyx (dg). Fig. 10. 'The section shows elongated cytopharynx (cyt). Fig. II. Microtubular prism (mp), spur (s) and band of microtubules (bm) pass along the cytopharynx. A symbiotic bacterium (sb) and system of pellicular microtubules (psm) are seen. Fig. 12. 'The spur (s) lies along the cytopharynx. The cytopharynx opening is surrounded with thin hairs (th). Fig. 13. Band of microtubules (bm) passes along the wall of the flagellar pocket (fp), thcn it bends (arrow) and continues along the cytopharynx (cyt). Fig. 14. In a large mitochondrion (m) there are osmiophilic bodies (ob); lamellar cristae (c) are scattered in the peripheral part of the organelle. The envelope of the nucleus (n) shows nuclear pores. Fig. 15. The section shows the mitochondrion (m) filled with fibrous material (fm). Fig. 16. Longitudinal section through mucocysts (mc). Nearby, a profile of the mitochondrion (m). Fig. 17. The Golgi apparatus (Ga) secretes vesicles (v) which give rise to mucocysts. Fig. 18. Extruded mucocysts have reticulate walls.
Ultrastructure of Phyllomitu8 apiculatu8
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Ultrastructure of Phyllomitu8 apiculatu8
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A. P.
MYLNIKOV
not been observed inside them. This can be explained by the fact that Phyllomitus was fixed at stationary growth phase and was not feeding. It has been impossible to observe the moment of food ingestion on sections. The nucleus, 1.8-2.2 pm in diameter, is situated near the flagellar kinetosomes. The nucleolus consists of several lobes, and the peripheral lobes surround the central part of the nucleolus (Figs. 2 and 14). The chromatin is inconspicuous. It is possible, however, the peripheral lobes consist of the chromatin. The nuclear pores can be noticed on several sections (Fig. 14). No stages of mitosis have been observed. The whole cell of the flagellate is permeated by a mitochondrion with lamellar cristae. The maximum length of the mitochondrion is 5-6 pm, the width being 2 pm. Besides the cristae, osmiophilic bodies and matrix are observed in the mitochondrion (Figs. 14-16). A typical kinetoplast has not been found. However, parts of the mitochondrion with condensed fibrous material resembling DNA have been noted on a number of sections (Fig. 15). Usually such kinetoplast-like structures are found in degraded or dead individuals. The diameter of this structure is equal to 1.5-2.0 pm. It has not been possible to detect mitochondrial DNA using FEULGEN'S method. Usually one mitochondrial profile is seen on sections of the anterior body end, and several profiles at the rear end. It is possible that the mitochondrion is branching near the rear end. At the anterior end there are peculiar trichocyst-like structures, here called mucocysts (according to HovAssE and MIGNOT 1975). The mucocysts are usually arranged in rows near the cytopharynx and in the lip (Figs. 2 and 8). There are 7-11 mucocysts in one row. Under the light microscope the mucocysts are seen after staining with haematoxylin or when using interference optics. A mucocyst consists of an outer tube, 120-150 nm in diameter and 2.5 pm long, and an inner amorphous tube 100 mn in diameter. A thread is seen inside the mucocyst. Between the tubes there is a bright zone (Figs. 16 and 17). It has not been possible to observe the process of liberation of the mucocysts. However, numerous extruded mucocysts have been fixed near the cells. They consist of a reticulate tube without inner contents (Fig. 18). The mesh size of the reticulum is 25 nm. The diameter of the reticulate tube is 150-200 nm. Thus, the diameter of the mucocysts increases after the liberation. The mucocysts are formed in the cisternae of the Golgi apparatus (Fig. 17). Extruded mucocysts are often seen inside the food vacuoles. Within the cells there are also symbiotic bacteria, 450 nm long and 300 nm in diameter (Fig. 11). It has been possible to observe division of these bacteria in the cytoplasm.
Discussion The following distinguishing features of the fine structure of Phyllomitus have been revealed in this work: paraxial rods in the flagella, subpellicular system of microtubules, three bands of kinetosome-associated microtubules, giant mitochondrion with lamellar cristae, tubular cytopharynx. All these features bring our species closer to the free-living bodonids whose ultrastructure has already been studied. This is primarily the case with Bodo caudatus, B. saltans (BROOKER 1971), B. curvifilus (BURZELL 1975), B. designis (EYDEN 1977), Pleuromonas jaculans (ANIKUSHIN 1975; KARPOV and ZHUKOV 1983) and also Rhynchomonas nasuta (SWALE 1973). Our investigations have revealed in Phyllomitus a layer of glycocalyx found in trypanosomes at certain stages of the life cycle (VICKERMAN and PRESTON 1976). This layer covers the flagella as well. It is believed to protect the trypanosomes from the host's antibodies. Its function in our predator is not clear.
Ultrastructure of Phyllomitu8 apiculatu8
SKUJA
1948
9
The free-living bodonids are characterized by the presence of a permanent cytopharynx which has the shape of a tube. Besides, it is covered with thin hairs similar to flagellar mastigonemes (KARPOV and ZHUKOV 1983). The same is observed in Phyllomitus. Like in the bodonids, the cytopharyngeal wall is reinforced in Phyllomitus by microtubules joined into bands consisting of 3, 5, and 6-7 microtubules. The last band is S-shaped. Practically the same kind of bands is known in the bodonids and cryptobiids (BRUGEROLLE et al. 1979; KARPOV and ZHUKOV 1983). The microtubular prism in Phyllomitus is similar to that in Bodo curvifilus and B. designis (BURZELL 1975; EVDEN 1977). The spur emerging from the kinetosome in Phyllomitus is also found in the euglenoid alga Isonema nigricans (SCHUSTER et al. 1968). EVDEN (1977) thinks that the microtubular prism is probably homologous to the band of 20 microtubules in Bodo spp. (BROOKER 1971) and to the quadritubular band in Cephalothamnium cyclopum (HITCHEN 1974). The flagellar pocket is the second depression on the Phyllomitus body. The heterodynamic flagella emerge from the bottom of the pocket. Such an arrangement of the flagella, i.e. parallel position of the kinetosomes, as well as the fact that the contractile vacuole discharges its contents into the flagellar pocket are characteristic of the kinetoplastids. The mastigonemes of Phyllomitus are similar to those in Pleuromonas jaculans (KARPOV and ZHUKOV 1983) but are less conspicuous than the mastigonemes in Bodo saltans and B. caudatus (BROOKER 1971). The nucleus, Golgi apparatus, contractile vacuole, and endoplasmic reticulum in Phyllomitus are typical of the kinetoplastids. One of the interesting features in Phyllomitus is the absence of a typical kinetoplast. Kinetoplast-like structures are found rarely and only in a few individuals. Probably such an organisation of the mitochondrion is more like that in the bodonid Ichthyobodo necator (JOVON and LOM 1969). Osmiophilic bodies in the mitochondrion of Phyllomitus are known to occur in the bodonid Spiromonas angusta (MACDoNALD and DARBVSHIRE 1977). The latter has other elements of morphology similar to those in Phyllomitus: heterodynamic flagella, cytopharynx with underlining microtubules, subpellicular microtubules, and a bundle of filaments near the cytopharynx opening. Moreover, the cytopharynx opening in S. angusta is situated at the anterior end of the body, like in Phyllomitus. Recently STEINBERG et al. (1983) noted stinging organelles at the anterior end of the body in Phyllomitus. Our electron micrographs show the structure of these organelles. Similar organelles are known in many Protozoa: ciliates, dinoflagellates, chloromonads, euglenomonadids (HAUSMANN 1978), but not in the bodonids. An especially great similarity in size and shape is observed between the mucocysts of Phyllomitus and those in euglenoid algae Isonema nigricans (SCHUSTER et al. 1968) and Entosiphon sulcatum (MIGNOT and HOVASSE 1973). The tubular wall of these organelles has a reticulate structure. They are filled with amorphous material. Somewhat larger mucocysts with a reticulate wall are found in Peranema trichophorum (HILENSKI and WALNE 1983). The function of the mucocysts in Phyllomitus is revealed still incompletely. The moment of extrusion of mucocysts has not been fixed by us. No prey stricken by a mucocysts has been observed. However, the presence of empty mucocysts in food vacuoles and the location of mature mucocysts close to the cytopharynx probably indicate participation of these organelles in food ingestion. Thus, our data have shown that the structure plan and the peculiarities of the ultrastructure of Phyllomitus allow to come to a conclusion that this species belongs to the suborder Bodonina of the order Kinetoplastida.
10
A. P. MYLNIKOV, Ultrastructure of Phyllomitu8 apiculatu8 SK1:"JA 1948
Literature ANIKUSHIN, N. F. (1975): Peculiarities of the ultrastructure of the mitochondrion in flagellates of the suborder Bodonina. Cytology 17: 94-95 (in Russian). BRUGEROLLE, G., LOM, J., NOHYNKOVA, E., et JOYON, L. (1979): Comparaison et evolution des structures cellulaires ches plusieurs especes de bodonides et cryptobiides appartenant aux genres Bodo, Cryptobia et Trypanopla8ma (Kinetoplastida, Mastigophora). Protistologica 15: 197-221. BROOKER, B. E. (1971): Fine structure of Bodo 8altan8 and Bodo caudatu8 (Zoomastigophorea: Protozoa) and their affinities with the Trypanosomatidae. Bull. Brit. Mus. (Natur. Hist.) Zoo!' 22: 87-102. BURZELL, Z. A. (1975): Fine structure of Bodo curvifilu8 GRIESS MANN (Kinetoplastida: Bodonidae). J. Protozoo!. 22: 35-39. EYDEN, B. P. (1977): Morphology and ultrastructure of Bodo de8igni8 SKUJA, 1948. Protistologica 13: 169-179. HAUSMANN, K. (1978): Extrusive organelles in protists. Int. Rev. Cyto!. 52: 197-276. HILENSKI, L. L., and W ALNE, P. L. (1983): Ultrastructure of mucocysts in Peranema trichophorum (Euglenophyceae). J. Protozoo!. 30: 491-496. HITCHEN, E. T. (1974): The fine structure of the colonial kinetoplastid flagellate Cephalothamnium cyclopum STEIN. J. Protozoo!. 21: 221-231. HOVASSE, R., et MIGNOT, J. P. (1975): Trichocystes et organites analogues ches les Protistes. Ann. Bio!. 14: 397-422. JOYON, L., et LOM, L. (1969): Etude cytologique, systematique et pathologique d'Ichthyobodo necator (HENNEGU¥, 1883) PINTO, 1928 (zooflagelle). J. Protozoo!. 16: 703-719. KARPOV, S. A., and ZHUKOV, B. F. (1983): The ultrathin structure of Pleuromona8 jaculan8 PERTY (Kinetoplastida, Zoomastigophorea, Sarcomastigophora). In: N. N. BANINA, T. V. BEYER and K. M. SUKHANOVA (eds.), Protozoa of activated sludge, Leningrad: 153-156 (in Russian). MAcDoNALD, C. M., and DARBYSHIRE, J. F. (1977): The morphology of a soil flagellate, Spiromona8 angu8ta (DuJ.) ALEXEIEFF (Mastigophorea: Protozoa). Protistologica 13: 441-450. MIGNOT, J. P., et HOVASSE, R. (1973): Nouvelle contribution a la connaissance des trichocystes: les organes grillages d'Ento8iphon 8ulcatum (Flagellata, Euglenida). Protistologica 9: 371-39l. :VIYLNIKOV, A. P. (1983): Feeding of predatory zooflagellates. BioI. vnutr. vod, Inform. bull. No. 60: 33-37 (in Russian). SCHUSTER, F. L., GOLDSTEIN, S., and HERSHENOV, B. (1968): Ultrastructure of a flagellate, 180nema nigrican8 nov. gen. nov. sp., from a polluted marine habitat. Protistologica 4: 141-149. SKUJA, N. (1948): Taxonomic des Phytoplanktons einiger Seen in Uppland, Schweden. Symb. Bot. Upsa!. 9/3: 1-399. STEINBERG, CH., LENHART, B., und KLEE, R. (1983): Bemerkungen zur Okologie cines farblosen Phytoflagellaten, Phyllomitu8 apiculatu8 SKUJA (1948), Cryptophyceae. Arch. Protistenkd. 127: 307-317. SWALE, E. 1\1. F. (1973): A study of the colourless flagellate Rhynchomona8 na8uta (STOKES) KLEBS. Bio!. J. Linn. Soc. 5: 255-264. VICKERJ\IAN, K. (1976): The diversity of the kinetoplastid flagellates. In: W. H. R. LUMSDEN and D. A. EVANS (cds.), Biology of kinetoplastida, pp. 1-34. London, New York, San Francisco. and PRESTON, T. 1\T. (1976): Comparative cell biology of kinetoplastid flagellates. Ibid. pp. 35-130. Author's address: A. P. MYLNIKOV, Laboratory of Biology of Lower Organisms, Institute of Biology of Inland Waters, USSR Academy of Sciences, 152742 Borok, Jaroslavl District, USSR.