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Comparative ultrastructure of the cytoskeleton and nucleus of Distigma (euglenozoa)
Europ. J. Protisto!. 35, 309-318 (1999) October 15, 1999 http://www.urbanfischer.de/journalslejp
European Journal of
PROTISTOLOGY
Comparative Ultrastructure of the Cytoskeleton and Nucleus of Distigma (Euglenozoa) David G.
Angeler1,:~,
Alexandra N. Mullner' and Michael Schagerll
1 Institute
of Plant Physiology, Department of Hydrobotany, University of Vienna, AlthanstraBe 14, A - 1090 Vienna, Austria 2 Institute of Botany, University of Vienna, Rennweg 14, A-1 030 Vienna, Austria
Summary Although the ultrastructure of the pellicle is currently considered as a valuable tool for systematic assessments within euglenoids, comparative aspects at the genus level are largely lacking. Whereas species of the pigmented genus Euglena have been the focus of some comparative ultrastructural analyses, colourless taxa have been generally neglected. This study attempts to describe the cytoarchitecture of the non-pigmented euglenoid genus Distigma, with special emphasis on the pellicle and nucleus morphology of D. curvatum Pringsheim, D. elegans Christen, D. glabrum Pringsheim ex Ettl nom. nud. non D. glabrum Christen, D. gracile Pringsheim, D. proteus var. proteus Ehrenberg emend. Pringsheim, D. proteus var. longicauda Angeler and D. sennii Pringsheim, All taxa possess the typical plastic type of pellicle which allows for euglenoid movements. Interspecific variations in pellicular strip construction may explain differences in form and degree of "metaboly". Based on the results the taxonomic significance of pellicle and nucleus ultrastructure in the genus can be discussed. Finally, some remarks on obviously mislabelled cultures are given. Key words: Distigma, Euglenozoa, Euglenophyta, Euglenida, Nucleus, Pellicle,Ultrastructure.
Introduction Pellicle ultrastructure, mitosis, feeding apparatus construction and architecture of the flagellar apparatus are considered important features for assessing phylogenetic relationships among members of the protistan group Euglenozoa [4], encompassing euglenoids, kine"corresponding author Present address: C.S.I.c., Centre for Environmental Sciences, Serrano 115 dpdo, E - 28006 Madrid, Spain; fax (0034) 91 5640800, e-mail [email protected] © 1999 by Urban & FischerVerlag
toplastids, diplonemids and the monotypic genus Postgaardi [32, 39, 40]. Whereas these features are of value at higher taxonomic levels, data on pellicle construction might also be of importance at lower taxonomic ranks within euglenoids [3, 19]. During the last decades, a number of ultrastructure studies and biochemical investigations have significantly contributed to the characterization of the cytoskeletal cortex in euglenoids (summarized in Dubreuil et a1. [10]). It is well established that a close relationship exists between strip architecture and the ability for and degree of euglenoid movement. Two types of pellicle can be distinguished: (1) a plastic type, allowing for changes in cell shape, so-called "metaboly" or euglenoid movement and (2) an aplastic type which is found in rigid taxa (e.g. Phacus, Sphenomonas). Transitions from high degree of metaboly to rigidity are evident even within a single genus, such as Euglena [9]. Considering the paucity of systematically informative structures at the genus level in euglenoids, especially in colourless taxa, Distigma Ehrenberg emend. Pringsheim was chosen for comparison of ultrastructure with traditional morphological features (length of flagella, location of the canalopening, cell shape and size, degree of euglenoid movement). Major taxonomic studies on Distigma are largely based on light microscope observations [5,6,27,28,33,34,35]. Cytological studies [13, 14, 16, 18, 24] and ultrastructural data [1, 11,20,21,22,24,42] are scarce and restricted to D. proteus Ehrenberg emend. Pringsheim. Only a few species have been compared considering the structure of the chondriome [d. 30], and comparative electron microscopic investigations are lacking. The main aim of this paper is to give a comparative presentation of the ultrastructure of various distigmids, and to elucidate correlations between fine structural features and light microscopical data, thereby dis0932-4739/99/35/03-309 $ 12.00/0
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cussing the value of pellicle and nucleus architecture for taxonomic purposes within the genus.
Materials and Methods Unialgal strains obtained from the SAG collection [31] were cultured in biphasic soil-water medium [29]. The strains were stored in the dark at 20 ± 2 "C. Cultures included in this study were D. proteus var. proteus (SAG B 1204-26a), D. proteus var. longicauda (SAG B 1216-6, listed as D. levis Pringsheim ex Ettl nom. nud. and re-described by Angeler [1]), D. sennii (SAG B 222.80), D. elegans (SAG B 224.80), D. curvatum (SAG B 1216-1b, SAG B 1204-29, SAG B 225.80), D. gracile (SAG B 216.80) and D. glabrum (SAG B 1216-5). Cells for SEM and CTEM were fixed in a mixture of 1 ml 25% (v/v) glutaraldehyde, 9 ml 0.1% (w/v) Na-cacodylate buffer, and 0.5 ml 4% (w/v) OS04 which has proven fruitful in preventing artificial induction of euglenoid movement [1], which frequently occurs by adding fixatives. Further details on ultrastructure methods and electron microscopy can be found in Angeler [1]. Data for strip width, number and inclination were obtained from at least 20 cells. Terminology of euglenoid cell surface structures largely follows the recommendations of Preisig et al. [26].
Results
Scanning Electron Microscopy (SEM) Distigma curvatum and D. glabrum
Figures 1-4 show cell shapes during swimming and euglenoid movement. The pellicle, composed of individual, non-ramified helically arranged strips, is shown in Fig. 2. The cell surface is covered to a varying extent with a layer of organic material, which sometimes renders it difficult to estimate the number of pellicular strips (Fig. 4). The angles of inclination of the strips vary depending on the degree of deformations (Fig. 1, 3). Whereas angles of about 75° relative to the cell length axis can be determined in the anterior part and in cytoplasmic dilatations, values decrease continuously to about 25° toward the posterior part of the cell. Also the width of single strips depends largely on the degree of deformation. In swimming cells, strip width is about 0.8 pm on the convex side versus 0.3 pm on the contracted concave face. Distigma proteus 5.1. and D. gracile
The number and orientation of the strips is constant as in other Distigma species, and is nearly identical in D. gracile, D. proteus var. proteus and D. proteus var. longicauda (Fig. 8, 9). The varieties of D. proteus possess around 18 strips, but are distinguished on the
Fig. 1-14. SEM micrographs of Distigma species. Scale bars: 1,4-14 = 10 Jlm;2, 3 = 8 Jlm. 1. Cell shape of D. curvatum during swimming. 2. D. curvatum undergoing euglenoid movement. Note the strip width on the concave part in relation to the convex face. 3. Metaboly stage of D. curvatum showing orientation of strips. 4. Metaboly stage of D. curvatum. Note the covering of the cell surface with a layer of organic material.
Comparative ultrastructure of Distigma
length ratio of the anterior and the posterior part of the cell (Fig. 5, 6). In non-distorted cells 8-9 strips are present in 10 pm. A single strip is about 1.2 pm broad. Inclinations of strips in free swimming cells are between 60-75° in the anterior region. Strips run nearly parallel
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to the cell length axis in the slender posterior part. As in D. curvatum and D. glabrum, strips appear fused in certain regions (Fig. 7) due to the excretion of mucilage. The strain of D. gracile could not be distinguished from D. proteus var. proteus.
5. Cell shape of D. proteus var. longicauda. 6. Cell shape of D. proteus var. proteus. 7. Metaboly stage of D. proteus var. proteus as viewed from the anterior pole. Strips appear fused in certain regions due to the excretion of mucilage. 8. Cell shape of D. proteus var. proteus, showing the constant number of strips throughout the entire cell. 9. Metaboly stage of D. proteus var. longicauda showing the constancy of strip number at the posterior pole.
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10. Cell shape of D. sennii. Note the relative length of the flagella. 11. Cell shape of D. elegans. 12. D. elegans undergoing euglenoid movement in the form of screw-like distortionand flattening. 13. Detail of the anterior pole of D. sennii showing nonbifurcating strips. 14. Detail of the posterior pole of D. senniishowing non-bifurcating strips.
Distigma sennii and D. eJegans The shapes of the monads as well as the orientation of the non-bifurcated strips are seen in Fig. 10-12. Details of the anterior and posterior ends are shown in Fig. 13 and 14. The width of a single strip is nearly constant throughout the entire cell surface of non-distorted cells (ca. 1.2 prn). 8-9 strips are present in 10 pm. The inclination of the strips is largely constant. Values of 51°-57°in the anterior third of the cell, 62°-700in the central part and 41 0 - 4 8 0 in the posterior cell third relative to the length axis were determined. Distigma sennii and D. elegans are distinguished on cell shapes and the higher degree of euglenoid movement in the latter (Fig. 12).
Conventional Transmission Electron Microscopy (CTEM) Pellicle Transverse sections of Distigma pellicular complexes show parallel strips that overlap along their lateral margins. The strip units form regular folds alternating with grooves. The plasma membrane (7-10 nm) with its trilamellar aspect is continuous along the ridges and grooves and is associated with a layer delineating the presumed protein part of the pellicle. Endoplasmic reticulum is closely associated with each strip unit (Fig . 19, 20) and forms the muciferous bodies. Each strip is subtended by a number of pellicular microtubules
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19
Fig. 15-26. CTEM micrographs of Distigma species. Scale bars: 15-21,24-26 = l prn; 22, 23 = 21lm. 15. Transverse section through the pellicle of D. curvatum, showing the extracellular sheet of organic material (EX). 16. Transverse section through the pellicle of D. proteus, showing serial arrangement of microtubules (arrows) and lacunae of endoplasmic reticulum (ER). 17. Pellicle cross section of D. elegans, showing lacunae of endoplasmic reticulum (ER), asymmetrical protein strips and two groups/types of microtubules. One group lies beneath the grooves (arrows), the second forms a continuation of the pellicular strip (arrowhead). 18. Pellicle cross section of D. sennii. This species differs from D. elegans by a single microtubule lining each groove. Extracellular stratified material is also present (EX). 19. Longitudinal section of the pellicle of D. sennii. Lacunae of endoplasmic reticulum form the muciferous bodies. 20. Muciferous body of D . sennii discharging its content. 21. Longitudinal section through the truncate anterior cell part of D. sennii. Pellicular micro tubules extend into the pericanalar region.
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which extend into the pericanalar region (Fig. 21). However, the number varies from one strip to the next. Based on their appearance in transverse sections three groups can be distinguished in Distigma:
1. D istigma curuatum and D. glabrum Transverse sections of the pellicle complex (Fig. 15) indicate that the more or less flat strips are covered with a ca. 120 nm thick layer of mucilage. Each strip shows
22. Cross section of the nucleus in D. curuatum, A single endosome with heterogeneous electron dens ity is present. Chromatin patches adhere to the inner nuclear membrane. The outer membrane is highly undulated. 23. Transverse section through the nucleus of D. proteus var.longicauda. Three endosome-like structures (E1-E3) are visible within the nucleoplasm. 24. Detail of an endosome in D. proteus. Endosomal structures partly surround each other. Fig.25-26. Details of endosomes in D. sennii and D. elegans, showing Pars amorpha (PA), Pars filamentosa (PF) and Pars granulosa (PG).
Comparative ultrastructure of Distigma
the same symmetry, with the origin of the protein layer close to the groove of two neighbouring strips. It extends continuously and equally in thickness along the plasmalemma. A set of 2-4 micro tubules underlines each strip, always close to the edge of the strip. 2. Distigma proteus s.l. and D. gracile
The CTEM re-examination of D. proteus is in good agreement with other studies [11,20, 21, 22, 42J. Data on pellicle construction is therefore summarized briefly: Distigma proteus var, proteus, D. proteus var. longicauda and also D. gracile show typical sigmoidal strips (Fig. 16), in contrast to D. curvatum and D. glabrum. A thin layer of mucilage covers the plasma membrane only. Beneath the plasma membrane is a faint electron dense proteinaceous layer (9-10 nm). It seems to be interrupted in the grooves. A group of 4-6 microtubules subtends one side of the strip whereas on the opposite side three microtubules are visible, one near the groove and the other two in the overhang. Lacunae of endoplasmic reticulum are located within each strip. 3. Distigma sennii and D. elegans
Transverse sections show typical flat outlines of the strips (Fig. 17, 18), somewhat like in D. curvatum and D. glabrum. Strips are individually covered with a stratified extra-cellular layer that is interrupted in the groove region. The protein layer below the plasma membrane is asymmetrical. It originates in the shallow groove region, increases in thickness and forms a hook or a short spine under the adjacent strip. A row of about four tubular structures extends from the hook. These differ in diameter from the microtubules beneath the grooves. Whereas a single cortical microtubular strand is found in D. sennii (Fig. 18), D. elegans possesses a set of 3-4 (Fig. 17). Nucleus
In undisturbed swimming monads , the nucleus is almost always found in the centre of the cell. Two unit membranes, the outer mostl y being highly undulated, typicall y surround it. A single endosome is visible in cross sections of D. curvatum, D. glabrum, D. sennii and D. elegans (Fig. 22, 25, 26), whereas 2 - 3 such structures are found in the varieties of D. proteus and the strain labelled D. gracile (Fig. 23, 24). However, the units marked as El, E2 and E3 in Figure 23 are not structurally uniform. In comparison with E3, El and E2 are similar in their heterogeneous electron density patterns and their less clear borders. In taxa containing . multiple endosomes, lamellar-like extensions some-
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times seem to extend from one (El ) to surround partly another one (E2; Fig. 24). In species with a single endosome, highly variable structures and outlines, sometimes containing satellite-like appendages, are found (Fig. 26). Endosomal structures are referred to as the (1) Pars amorpha comprising the fine granular matrix [41J, (2) Pars granulosa, an electron dense layer located at the periphery of the endosomes and (3) Pars filamentosa, the electron transparent region within the matrix (Fig. 25). Patches of condensed chromatin are distributed throughout the nucleoplasm and adhere to the inner nuclear membrane (Fig. 22, 23).
Discussion This study aimed at a comparison of the cytoarchitecture of Distigma species, putting special emphasis on pellicle and nucleus construction. Unfortunately, only a limited number of strains was available for this study owing to paucity of cultures and an infrequent occurrence in nature. For D. elegans and D. sennii our conclusions are based on observations from single cultures. However, for species for which more isolates were available, e.g. D. curvatum, D. proteus var. proteus and D. proteus var.longicauda, no substantial variation can be found between strains [1, this study], We therefore assume that further isolates of D. elegans and D. sennii will not show substantial differences. All distigmids observed so far possess a typical plastic type of pellicle. It is composed of numerous helically arranged non-ramified strips, their number being constant throughout the entire cell. This is not a general characteristic of euglenoids with a plastic cytoskeleton, as reduction of strip number has been shown in the anterior and posterior part of non-dividing cells of Euglena pisciformis [17, 18J, E. gracilis [15J, E. oxyuris var. minor [2J, Eutreptia pertyi [7J, A stasia longa [38J and members of the genus Khawkinea (D.G. Angeler, unpublished results). The systematic significance of constant versus decreasing strip number currently cannot be estimated due to paucity of inter- and intrageneric data. It has been shown for various euglenoid flagellates that discontinuous periodic structural elements, socalled "plate-like projections", associate with the protein matrix subtending the plasmalemma. For Euglena, Dragos et al. [9J discussed a correlation with clearly evident "plate-like projections" and low degree of euglenoid movement and vice versa, i.e. small or absent projections and pronounced metaboly. Paradoxically, Euglena ehrenbergii is a highly metabolic species but possesses very prominent "plate-like projections" [37J. Nevertheless, such structures were absent in the strips of all Distigma species examined. Since all distigmids
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except D. sennii show a high degree of euglenoid movement, other mechanisms may account for the differences in the degree of euglenoid movement in the genus. Current hypotheses on euglenoid movement [11, 12, 24, 36] claim mechanical procedures to be responsible for cell contractions, as known from the sliding filament theory. Investigations of Murray [23, 24] demonstrating microtubule-plasma membrane links in D. proteus and ATPases located between the cortical microtubules and the plasmalemma agree with the studies of Dentler et al. [8], which propose structural connections between the ciliary membrane and axonemal microtubules in Tetrahymena. Newton [25] suggested such complexes to be responsible for plasma membrane-microtubule sliding in the non-flagellated spermatozoon of the turbellarian Macrostomum tubum. Distigma sennii and D. elegans share nearly identical strip constructions but are distinguished in the number of micro tubules lining the strips. This difference might explain why both species exhibit varying degrees of euglenoid movement while D. elegans exhibits marked euglenoid movement and D. sennii appears nearly rigid under undisturbed swimming conditions. The former may possess more microtubule-membrane links [24] and additional microtubule-microtubule connections [12] than D. sennii. Distigma proteus, D. gracile, D. glabrum and D. curuatum also possess more than one microtubular strand beneath each strip and show the same extent as in D. elegans (Fig. 27). Further studies are needed to confirm whether there is a closer correlation in euglenoids between the number of microtubules and the extent of metaboly. Whereas D. proteus, D. gracile, D. glabrum and D. curvatum almost only form cytoplasmic dilatations that pass along the length of the cell during euglenoid movement, metaboly in D. sennii and D. elegans is predominately characterized by flattenings and screw-like distortions. Cytoplasmic swellings also appear, especially in D. elegans. Deviations in pellicle construction, maybe unequal symmetry of the protein layer, may explain these differences. Mignot [20] and Bourrelly et al.
number of cortical microtubules
6 D. sennii
D. CUNatum D. elegans D. proteus 5.1 .
high low----~-~-~IIIII!I!---~-_· degree of euglenoid movement
Fig. 27. Correlation between the number of cortical microtubules and the degree of euglenoid movement in Distigma species used in this study.
[2] have discussed a relationship between the protein layer and the extent of euglenoid movement: Based on the paucity of stable diagnostic criteria available for colourless osmotrophic euglenoids (length of flagella, position of the canal opening etc.), ultrastructure data may be considered for systematic assessments. Since features such as mitosis and construction of the flagellar apparatus seem to vary insignificantly at lower taxonomic levels, pellicle ultrastructure remains one promising systematic tool because it allows us to distinguish even at the species level. In a previous report, Angeler [1] stressed the idea to unite Distigma strains with identical ultrastructure into a single species but to consider deviations in cell shape as infraspecific features. Hence, the variety Distigma proteus var. longicauda was established to include invalid species [1]. Besides pellicular features, the number of endosome-like structures in the nucleoplasm proved to be a promising systematic tool in Distigma. All strains now assigned to the D. proteus group were shown to possess multiple endosome-like structures. This result was in good agreement with data on D. proteus from Hollande [13, 14], Lackey [16], Leedale [17] and Mignot [22]. However, endosome construction in D. proteus has not been clear classified yet, since structural differences occur within single units. Based on the similarity in electron density pattern of El and E2 (Fig. 23) and. the partly surrounding lamellar extensions (Fig. 24), they might constitute a single highly lobed complex rather than two separate units. The total separation of E1/E2 from E3 has been \shown [1]. Further investigations shall clarify the structure of these endosome units in relation to ontogenetic shifts. Additional ultrastructural data obtained in this study allow speculation about relationships within Distigma. If phenetic data represents phylogenetic relatedness, several groups can be distinguished based upon vegetative morphology. It is, however, beyond the scope of this paper to considersubgeneric classifications because of absence of data from other species and support from morphologically independent markers, e.g. of molecular nature. Molecular phylogenetic analyses shall resolve evolutionary relatedness among the distigmids, and also their relationships to other colourless and pigmented taxa. Group 1 comprises D. proteus s.1., group 2 D. curuatum and group 3 contains D. sennii and D. elegans (characters listed in Tab. 1). Whereas group 1 can be clearly distinguished from groups 2 and 3, the latter share some similarities: the short ventral flagellum (length ca. 4 pm), the single endosome and similar pellicle architecture. Clear differences are the symmetry of the protein layer as well as the form of the anterior pole in swimming cells. In group 3, D . sennii may be derived from D. elegans on loss of cortical microtubules.
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Table 1. Delineation of three groups within Distigma based on morphological characteristics. D. proteus group
D. curuatum group
D. sennii group
D. curuaturn
Shape of anterior end Swimming movement Length of long flagellum Length of short flagellum Nuclear endosomes Pellicular striae Protein layer Cortical micro tubules
D. proteus var, proteus D. proteus var, longicauda round rotating, trembling ca. 35 urn (-2/3 cell length) ca. 13 prn ( 1/3 cell length) 2-3 sigmoidal outlines even thickness 4-6
round rotating ca. 25 \lm (-2/3 cell length) ca. 4-5 prn 1 flat-slightly convex even thickness 2-4
D. sennii D. elegans truncate rotating ca. 25 urn (- 2/3 cell length) ca. 4-5 urn
Euglenoid movement
pronounced
pronounced
Taxa included
The present study has contributed to the identification of obviously mislabelled cultures. In our re-examination, the strain labelled D. gracile (SAG B 216.80) was shown to correspond in all diagnostic characters to D. proteus var. proteus, both in the light microscope as well as at the fine structure level, rather than being in agreement with Pringsheim's light-microscopical description [28]. The second strain, D. glabrum (SAG B 1216-5), was initially proposed to be a new species by Pringsheim, whose idea was later substantiated by Ettl, but a valid description was never published (see [31]). The suggested epithet would be a homonym for D. glabrum, though, a species originally described by Christen [5] . Re-examination of this strain did not reveal sufficient evidence to lend support for the erection of a new species. The morphological characteristics were typical of D. curvatum. Both strains might have suffered from mistransfer. The strain D. gracile (SAG B216.80) should therefore be assigned to D. proteus var. proteus, and D. glabrum (SAG B 1216-5) to D. curvatum. Acknowledgements: The authors wish to express their gratitude to Prof. Dr. UG Schlosser (Gottingen) for his generous gift of strains. This study was financially supported by a research grant from the University of Vienna.
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parente entre Astasiacees et Euglenacees. Arch. Zool. expo gen. 86, 499-549. Schlosser U.G. (1994): SAG-Sammlung von Algenkulturen at the University of Gottingen. Catalogue of strains 1994. Bot. Acta 107, 111-186. Simpson A.G.B. (1997): The identity and composition of the Euglenozoa. Arch. Protistenkd. 148,318-328. Skuja H. (1939) Beitrage zur Algenflora Lettlands II. Acta Hort. Bot. Univ. Latv, 11/12,41-169. Skuja H. (1948): Taxonomie des Phytoplanktons einiger Seen in Uppland, Schweden. Symb. Bot. Upsal. 9, 1-399. Skuja H (1956): Taxonomische und biologische Studien iiber das Phytoplankton schwedischer Binnengewasser, Nova Acta Reg. Soc. Sci. Upsal. Ser. IV, 16 (3),1-404. Suzaki T. and Williamson R.E. (1985): Euglenoid movement in Euglena fusca: evidence for sliding between pellicular strips. Protoplasma 124, 137-146. Suzaki T. and Williamson R.E. (1986): Pellicular ultrastructure and euglenoid movement in Euglena ehrenbergii Klebs and Euglena oxyuris Schmarda. J. Protozoo I. 33,165-171. Suzaki T. and Williamson R.E. (1986): Ultrastructure and sliding of pellicular structures during euglenoid movement in Astasia tonga Pringsheim (Sarcomastigophora, Euglenida).J. Protozool. 33,179-184. Triemer R.E. and Farmer M.A. (1991a): An ultrastructural comparison of the mitotic apparatus, feeding apparatus, flagellar apparatus and cytoskeleton in euglenoids and kinetoplasrids. Protoplasma 164,91-104. Triemer R.E. and Farmer M.A. (1991b): The ultrastructural organization of the heterotrophic euglenids and its evolutionary implications. In: Patterson D.J. and Larsen]. (eds.): The biology of free-living heterotrophic flagellates, pp. 185-204. Clarendon Press, Oxford. Ude J. and Koch M. (1994): Die Zelle, Atlas der Ultrastrukrur, G. Fischer, Jena, Stuttgart. Yamaguchi T. and Anderson O.R. (1994): Fine structure of laboratory cultured Distigma proteus and cytochemical localization of acid phosphatase. J. Morphol. 219, 89-99 .
European Journal of
PROTISTOLOGY
Comparative Ultrastructure of the Cytoskeleton and Nucleus of Distigma (Euglenozoa) David G.
Angeler1,:~,
Alexandra N. Mullner' and Michael Schagerll
1 Institute
of Plant Physiology, Department of Hydrobotany, University of Vienna, AlthanstraBe 14, A - 1090 Vienna, Austria 2 Institute of Botany, University of Vienna, Rennweg 14, A-1 030 Vienna, Austria
Summary Although the ultrastructure of the pellicle is currently considered as a valuable tool for systematic assessments within euglenoids, comparative aspects at the genus level are largely lacking. Whereas species of the pigmented genus Euglena have been the focus of some comparative ultrastructural analyses, colourless taxa have been generally neglected. This study attempts to describe the cytoarchitecture of the non-pigmented euglenoid genus Distigma, with special emphasis on the pellicle and nucleus morphology of D. curvatum Pringsheim, D. elegans Christen, D. glabrum Pringsheim ex Ettl nom. nud. non D. glabrum Christen, D. gracile Pringsheim, D. proteus var. proteus Ehrenberg emend. Pringsheim, D. proteus var. longicauda Angeler and D. sennii Pringsheim, All taxa possess the typical plastic type of pellicle which allows for euglenoid movements. Interspecific variations in pellicular strip construction may explain differences in form and degree of "metaboly". Based on the results the taxonomic significance of pellicle and nucleus ultrastructure in the genus can be discussed. Finally, some remarks on obviously mislabelled cultures are given. Key words: Distigma, Euglenozoa, Euglenophyta, Euglenida, Nucleus, Pellicle,Ultrastructure.
Introduction Pellicle ultrastructure, mitosis, feeding apparatus construction and architecture of the flagellar apparatus are considered important features for assessing phylogenetic relationships among members of the protistan group Euglenozoa [4], encompassing euglenoids, kine"corresponding author Present address: C.S.I.c., Centre for Environmental Sciences, Serrano 115 dpdo, E - 28006 Madrid, Spain; fax (0034) 91 5640800, e-mail [email protected] © 1999 by Urban & FischerVerlag
toplastids, diplonemids and the monotypic genus Postgaardi [32, 39, 40]. Whereas these features are of value at higher taxonomic levels, data on pellicle construction might also be of importance at lower taxonomic ranks within euglenoids [3, 19]. During the last decades, a number of ultrastructure studies and biochemical investigations have significantly contributed to the characterization of the cytoskeletal cortex in euglenoids (summarized in Dubreuil et a1. [10]). It is well established that a close relationship exists between strip architecture and the ability for and degree of euglenoid movement. Two types of pellicle can be distinguished: (1) a plastic type, allowing for changes in cell shape, so-called "metaboly" or euglenoid movement and (2) an aplastic type which is found in rigid taxa (e.g. Phacus, Sphenomonas). Transitions from high degree of metaboly to rigidity are evident even within a single genus, such as Euglena [9]. Considering the paucity of systematically informative structures at the genus level in euglenoids, especially in colourless taxa, Distigma Ehrenberg emend. Pringsheim was chosen for comparison of ultrastructure with traditional morphological features (length of flagella, location of the canalopening, cell shape and size, degree of euglenoid movement). Major taxonomic studies on Distigma are largely based on light microscope observations [5,6,27,28,33,34,35]. Cytological studies [13, 14, 16, 18, 24] and ultrastructural data [1, 11,20,21,22,24,42] are scarce and restricted to D. proteus Ehrenberg emend. Pringsheim. Only a few species have been compared considering the structure of the chondriome [d. 30], and comparative electron microscopic investigations are lacking. The main aim of this paper is to give a comparative presentation of the ultrastructure of various distigmids, and to elucidate correlations between fine structural features and light microscopical data, thereby dis0932-4739/99/35/03-309 $ 12.00/0
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cussing the value of pellicle and nucleus architecture for taxonomic purposes within the genus.
Materials and Methods Unialgal strains obtained from the SAG collection [31] were cultured in biphasic soil-water medium [29]. The strains were stored in the dark at 20 ± 2 "C. Cultures included in this study were D. proteus var. proteus (SAG B 1204-26a), D. proteus var. longicauda (SAG B 1216-6, listed as D. levis Pringsheim ex Ettl nom. nud. and re-described by Angeler [1]), D. sennii (SAG B 222.80), D. elegans (SAG B 224.80), D. curvatum (SAG B 1216-1b, SAG B 1204-29, SAG B 225.80), D. gracile (SAG B 216.80) and D. glabrum (SAG B 1216-5). Cells for SEM and CTEM were fixed in a mixture of 1 ml 25% (v/v) glutaraldehyde, 9 ml 0.1% (w/v) Na-cacodylate buffer, and 0.5 ml 4% (w/v) OS04 which has proven fruitful in preventing artificial induction of euglenoid movement [1], which frequently occurs by adding fixatives. Further details on ultrastructure methods and electron microscopy can be found in Angeler [1]. Data for strip width, number and inclination were obtained from at least 20 cells. Terminology of euglenoid cell surface structures largely follows the recommendations of Preisig et al. [26].
Results
Scanning Electron Microscopy (SEM) Distigma curvatum and D. glabrum
Figures 1-4 show cell shapes during swimming and euglenoid movement. The pellicle, composed of individual, non-ramified helically arranged strips, is shown in Fig. 2. The cell surface is covered to a varying extent with a layer of organic material, which sometimes renders it difficult to estimate the number of pellicular strips (Fig. 4). The angles of inclination of the strips vary depending on the degree of deformations (Fig. 1, 3). Whereas angles of about 75° relative to the cell length axis can be determined in the anterior part and in cytoplasmic dilatations, values decrease continuously to about 25° toward the posterior part of the cell. Also the width of single strips depends largely on the degree of deformation. In swimming cells, strip width is about 0.8 pm on the convex side versus 0.3 pm on the contracted concave face. Distigma proteus 5.1. and D. gracile
The number and orientation of the strips is constant as in other Distigma species, and is nearly identical in D. gracile, D. proteus var. proteus and D. proteus var. longicauda (Fig. 8, 9). The varieties of D. proteus possess around 18 strips, but are distinguished on the
Fig. 1-14. SEM micrographs of Distigma species. Scale bars: 1,4-14 = 10 Jlm;2, 3 = 8 Jlm. 1. Cell shape of D. curvatum during swimming. 2. D. curvatum undergoing euglenoid movement. Note the strip width on the concave part in relation to the convex face. 3. Metaboly stage of D. curvatum showing orientation of strips. 4. Metaboly stage of D. curvatum. Note the covering of the cell surface with a layer of organic material.
Comparative ultrastructure of Distigma
length ratio of the anterior and the posterior part of the cell (Fig. 5, 6). In non-distorted cells 8-9 strips are present in 10 pm. A single strip is about 1.2 pm broad. Inclinations of strips in free swimming cells are between 60-75° in the anterior region. Strips run nearly parallel
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to the cell length axis in the slender posterior part. As in D. curvatum and D. glabrum, strips appear fused in certain regions (Fig. 7) due to the excretion of mucilage. The strain of D. gracile could not be distinguished from D. proteus var. proteus.
5. Cell shape of D. proteus var. longicauda. 6. Cell shape of D. proteus var. proteus. 7. Metaboly stage of D. proteus var. proteus as viewed from the anterior pole. Strips appear fused in certain regions due to the excretion of mucilage. 8. Cell shape of D. proteus var. proteus, showing the constant number of strips throughout the entire cell. 9. Metaboly stage of D. proteus var. longicauda showing the constancy of strip number at the posterior pole.
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10. Cell shape of D. sennii. Note the relative length of the flagella. 11. Cell shape of D. elegans. 12. D. elegans undergoing euglenoid movement in the form of screw-like distortionand flattening. 13. Detail of the anterior pole of D. sennii showing nonbifurcating strips. 14. Detail of the posterior pole of D. senniishowing non-bifurcating strips.
Distigma sennii and D. eJegans The shapes of the monads as well as the orientation of the non-bifurcated strips are seen in Fig. 10-12. Details of the anterior and posterior ends are shown in Fig. 13 and 14. The width of a single strip is nearly constant throughout the entire cell surface of non-distorted cells (ca. 1.2 prn). 8-9 strips are present in 10 pm. The inclination of the strips is largely constant. Values of 51°-57°in the anterior third of the cell, 62°-700in the central part and 41 0 - 4 8 0 in the posterior cell third relative to the length axis were determined. Distigma sennii and D. elegans are distinguished on cell shapes and the higher degree of euglenoid movement in the latter (Fig. 12).
Conventional Transmission Electron Microscopy (CTEM) Pellicle Transverse sections of Distigma pellicular complexes show parallel strips that overlap along their lateral margins. The strip units form regular folds alternating with grooves. The plasma membrane (7-10 nm) with its trilamellar aspect is continuous along the ridges and grooves and is associated with a layer delineating the presumed protein part of the pellicle. Endoplasmic reticulum is closely associated with each strip unit (Fig . 19, 20) and forms the muciferous bodies. Each strip is subtended by a number of pellicular microtubules
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19
Fig. 15-26. CTEM micrographs of Distigma species. Scale bars: 15-21,24-26 = l prn; 22, 23 = 21lm. 15. Transverse section through the pellicle of D. curvatum, showing the extracellular sheet of organic material (EX). 16. Transverse section through the pellicle of D. proteus, showing serial arrangement of microtubules (arrows) and lacunae of endoplasmic reticulum (ER). 17. Pellicle cross section of D. elegans, showing lacunae of endoplasmic reticulum (ER), asymmetrical protein strips and two groups/types of microtubules. One group lies beneath the grooves (arrows), the second forms a continuation of the pellicular strip (arrowhead). 18. Pellicle cross section of D. sennii. This species differs from D. elegans by a single microtubule lining each groove. Extracellular stratified material is also present (EX). 19. Longitudinal section of the pellicle of D. sennii. Lacunae of endoplasmic reticulum form the muciferous bodies. 20. Muciferous body of D . sennii discharging its content. 21. Longitudinal section through the truncate anterior cell part of D. sennii. Pellicular micro tubules extend into the pericanalar region.
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which extend into the pericanalar region (Fig. 21). However, the number varies from one strip to the next. Based on their appearance in transverse sections three groups can be distinguished in Distigma:
1. D istigma curuatum and D. glabrum Transverse sections of the pellicle complex (Fig. 15) indicate that the more or less flat strips are covered with a ca. 120 nm thick layer of mucilage. Each strip shows
22. Cross section of the nucleus in D. curuatum, A single endosome with heterogeneous electron dens ity is present. Chromatin patches adhere to the inner nuclear membrane. The outer membrane is highly undulated. 23. Transverse section through the nucleus of D. proteus var.longicauda. Three endosome-like structures (E1-E3) are visible within the nucleoplasm. 24. Detail of an endosome in D. proteus. Endosomal structures partly surround each other. Fig.25-26. Details of endosomes in D. sennii and D. elegans, showing Pars amorpha (PA), Pars filamentosa (PF) and Pars granulosa (PG).
Comparative ultrastructure of Distigma
the same symmetry, with the origin of the protein layer close to the groove of two neighbouring strips. It extends continuously and equally in thickness along the plasmalemma. A set of 2-4 micro tubules underlines each strip, always close to the edge of the strip. 2. Distigma proteus s.l. and D. gracile
The CTEM re-examination of D. proteus is in good agreement with other studies [11,20, 21, 22, 42J. Data on pellicle construction is therefore summarized briefly: Distigma proteus var, proteus, D. proteus var. longicauda and also D. gracile show typical sigmoidal strips (Fig. 16), in contrast to D. curvatum and D. glabrum. A thin layer of mucilage covers the plasma membrane only. Beneath the plasma membrane is a faint electron dense proteinaceous layer (9-10 nm). It seems to be interrupted in the grooves. A group of 4-6 microtubules subtends one side of the strip whereas on the opposite side three microtubules are visible, one near the groove and the other two in the overhang. Lacunae of endoplasmic reticulum are located within each strip. 3. Distigma sennii and D. elegans
Transverse sections show typical flat outlines of the strips (Fig. 17, 18), somewhat like in D. curvatum and D. glabrum. Strips are individually covered with a stratified extra-cellular layer that is interrupted in the groove region. The protein layer below the plasma membrane is asymmetrical. It originates in the shallow groove region, increases in thickness and forms a hook or a short spine under the adjacent strip. A row of about four tubular structures extends from the hook. These differ in diameter from the microtubules beneath the grooves. Whereas a single cortical microtubular strand is found in D. sennii (Fig. 18), D. elegans possesses a set of 3-4 (Fig. 17). Nucleus
In undisturbed swimming monads , the nucleus is almost always found in the centre of the cell. Two unit membranes, the outer mostl y being highly undulated, typicall y surround it. A single endosome is visible in cross sections of D. curvatum, D. glabrum, D. sennii and D. elegans (Fig. 22, 25, 26), whereas 2 - 3 such structures are found in the varieties of D. proteus and the strain labelled D. gracile (Fig. 23, 24). However, the units marked as El, E2 and E3 in Figure 23 are not structurally uniform. In comparison with E3, El and E2 are similar in their heterogeneous electron density patterns and their less clear borders. In taxa containing . multiple endosomes, lamellar-like extensions some-
315
times seem to extend from one (El ) to surround partly another one (E2; Fig. 24). In species with a single endosome, highly variable structures and outlines, sometimes containing satellite-like appendages, are found (Fig. 26). Endosomal structures are referred to as the (1) Pars amorpha comprising the fine granular matrix [41J, (2) Pars granulosa, an electron dense layer located at the periphery of the endosomes and (3) Pars filamentosa, the electron transparent region within the matrix (Fig. 25). Patches of condensed chromatin are distributed throughout the nucleoplasm and adhere to the inner nuclear membrane (Fig. 22, 23).
Discussion This study aimed at a comparison of the cytoarchitecture of Distigma species, putting special emphasis on pellicle and nucleus construction. Unfortunately, only a limited number of strains was available for this study owing to paucity of cultures and an infrequent occurrence in nature. For D. elegans and D. sennii our conclusions are based on observations from single cultures. However, for species for which more isolates were available, e.g. D. curvatum, D. proteus var. proteus and D. proteus var.longicauda, no substantial variation can be found between strains [1, this study], We therefore assume that further isolates of D. elegans and D. sennii will not show substantial differences. All distigmids observed so far possess a typical plastic type of pellicle. It is composed of numerous helically arranged non-ramified strips, their number being constant throughout the entire cell. This is not a general characteristic of euglenoids with a plastic cytoskeleton, as reduction of strip number has been shown in the anterior and posterior part of non-dividing cells of Euglena pisciformis [17, 18J, E. gracilis [15J, E. oxyuris var. minor [2J, Eutreptia pertyi [7J, A stasia longa [38J and members of the genus Khawkinea (D.G. Angeler, unpublished results). The systematic significance of constant versus decreasing strip number currently cannot be estimated due to paucity of inter- and intrageneric data. It has been shown for various euglenoid flagellates that discontinuous periodic structural elements, socalled "plate-like projections", associate with the protein matrix subtending the plasmalemma. For Euglena, Dragos et al. [9J discussed a correlation with clearly evident "plate-like projections" and low degree of euglenoid movement and vice versa, i.e. small or absent projections and pronounced metaboly. Paradoxically, Euglena ehrenbergii is a highly metabolic species but possesses very prominent "plate-like projections" [37J. Nevertheless, such structures were absent in the strips of all Distigma species examined. Since all distigmids
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except D. sennii show a high degree of euglenoid movement, other mechanisms may account for the differences in the degree of euglenoid movement in the genus. Current hypotheses on euglenoid movement [11, 12, 24, 36] claim mechanical procedures to be responsible for cell contractions, as known from the sliding filament theory. Investigations of Murray [23, 24] demonstrating microtubule-plasma membrane links in D. proteus and ATPases located between the cortical microtubules and the plasmalemma agree with the studies of Dentler et al. [8], which propose structural connections between the ciliary membrane and axonemal microtubules in Tetrahymena. Newton [25] suggested such complexes to be responsible for plasma membrane-microtubule sliding in the non-flagellated spermatozoon of the turbellarian Macrostomum tubum. Distigma sennii and D. elegans share nearly identical strip constructions but are distinguished in the number of micro tubules lining the strips. This difference might explain why both species exhibit varying degrees of euglenoid movement while D. elegans exhibits marked euglenoid movement and D. sennii appears nearly rigid under undisturbed swimming conditions. The former may possess more microtubule-membrane links [24] and additional microtubule-microtubule connections [12] than D. sennii. Distigma proteus, D. gracile, D. glabrum and D. curuatum also possess more than one microtubular strand beneath each strip and show the same extent as in D. elegans (Fig. 27). Further studies are needed to confirm whether there is a closer correlation in euglenoids between the number of microtubules and the extent of metaboly. Whereas D. proteus, D. gracile, D. glabrum and D. curvatum almost only form cytoplasmic dilatations that pass along the length of the cell during euglenoid movement, metaboly in D. sennii and D. elegans is predominately characterized by flattenings and screw-like distortions. Cytoplasmic swellings also appear, especially in D. elegans. Deviations in pellicle construction, maybe unequal symmetry of the protein layer, may explain these differences. Mignot [20] and Bourrelly et al.
number of cortical microtubules
6 D. sennii
D. CUNatum D. elegans D. proteus 5.1 .
high low----~-~-~IIIII!I!---~-_· degree of euglenoid movement
Fig. 27. Correlation between the number of cortical microtubules and the degree of euglenoid movement in Distigma species used in this study.
[2] have discussed a relationship between the protein layer and the extent of euglenoid movement: Based on the paucity of stable diagnostic criteria available for colourless osmotrophic euglenoids (length of flagella, position of the canal opening etc.), ultrastructure data may be considered for systematic assessments. Since features such as mitosis and construction of the flagellar apparatus seem to vary insignificantly at lower taxonomic levels, pellicle ultrastructure remains one promising systematic tool because it allows us to distinguish even at the species level. In a previous report, Angeler [1] stressed the idea to unite Distigma strains with identical ultrastructure into a single species but to consider deviations in cell shape as infraspecific features. Hence, the variety Distigma proteus var. longicauda was established to include invalid species [1]. Besides pellicular features, the number of endosome-like structures in the nucleoplasm proved to be a promising systematic tool in Distigma. All strains now assigned to the D. proteus group were shown to possess multiple endosome-like structures. This result was in good agreement with data on D. proteus from Hollande [13, 14], Lackey [16], Leedale [17] and Mignot [22]. However, endosome construction in D. proteus has not been clear classified yet, since structural differences occur within single units. Based on the similarity in electron density pattern of El and E2 (Fig. 23) and. the partly surrounding lamellar extensions (Fig. 24), they might constitute a single highly lobed complex rather than two separate units. The total separation of E1/E2 from E3 has been \shown [1]. Further investigations shall clarify the structure of these endosome units in relation to ontogenetic shifts. Additional ultrastructural data obtained in this study allow speculation about relationships within Distigma. If phenetic data represents phylogenetic relatedness, several groups can be distinguished based upon vegetative morphology. It is, however, beyond the scope of this paper to considersubgeneric classifications because of absence of data from other species and support from morphologically independent markers, e.g. of molecular nature. Molecular phylogenetic analyses shall resolve evolutionary relatedness among the distigmids, and also their relationships to other colourless and pigmented taxa. Group 1 comprises D. proteus s.1., group 2 D. curuatum and group 3 contains D. sennii and D. elegans (characters listed in Tab. 1). Whereas group 1 can be clearly distinguished from groups 2 and 3, the latter share some similarities: the short ventral flagellum (length ca. 4 pm), the single endosome and similar pellicle architecture. Clear differences are the symmetry of the protein layer as well as the form of the anterior pole in swimming cells. In group 3, D . sennii may be derived from D. elegans on loss of cortical microtubules.
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Table 1. Delineation of three groups within Distigma based on morphological characteristics. D. proteus group
D. curuatum group
D. sennii group
D. curuaturn
Shape of anterior end Swimming movement Length of long flagellum Length of short flagellum Nuclear endosomes Pellicular striae Protein layer Cortical micro tubules
D. proteus var, proteus D. proteus var, longicauda round rotating, trembling ca. 35 urn (-2/3 cell length) ca. 13 prn ( 1/3 cell length) 2-3 sigmoidal outlines even thickness 4-6
round rotating ca. 25 \lm (-2/3 cell length) ca. 4-5 prn 1 flat-slightly convex even thickness 2-4
D. sennii D. elegans truncate rotating ca. 25 urn (- 2/3 cell length) ca. 4-5 urn
Euglenoid movement
pronounced
pronounced
Taxa included
The present study has contributed to the identification of obviously mislabelled cultures. In our re-examination, the strain labelled D. gracile (SAG B 216.80) was shown to correspond in all diagnostic characters to D. proteus var. proteus, both in the light microscope as well as at the fine structure level, rather than being in agreement with Pringsheim's light-microscopical description [28]. The second strain, D. glabrum (SAG B 1216-5), was initially proposed to be a new species by Pringsheim, whose idea was later substantiated by Ettl, but a valid description was never published (see [31]). The suggested epithet would be a homonym for D. glabrum, though, a species originally described by Christen [5] . Re-examination of this strain did not reveal sufficient evidence to lend support for the erection of a new species. The morphological characteristics were typical of D. curvatum. Both strains might have suffered from mistransfer. The strain D. gracile (SAG B216.80) should therefore be assigned to D. proteus var. proteus, and D. glabrum (SAG B 1216-5) to D. curvatum. Acknowledgements: The authors wish to express their gratitude to Prof. Dr. UG Schlosser (Gottingen) for his generous gift of strains. This study was financially supported by a research grant from the University of Vienna.
References Angeler D.G. (1999): Distigma proteus var. longicauda var, nov. - a new colourless euglenoid described from cultures. Arch. Hydrobiol. Suppl. 127, Algolog. Stud. 92, 19-33. 2 Bourrelly P., Coute A. and Rino ].A. (1976): Ultrastructure de la cuticule de quelques eugleniens: 1. Euglena oxyuris var, minor Defl. et Euglena spirogyra var, fu sca (Klebs) Lemm. Protisrologica 12,623-628. 3 Cann J.P. (1986): Ultrastructural observations of taxonomic importance on the euglenoid genera Gyropaigne
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9 10
11 12 13 14 15 16 .
1
flat outlines uneven thickness D. sennii: 1 D. elegans: 3 - 4 D. sennii: slight D. elegans: pronounced
Skuja, Parmidium Christen, and Rhabdospira Pringshe im (Euglenida: Rhabdomonadina). Arch. Protistenkd. 132, 395-401. Cavalier-Smith T. (1981): Eukaryote kingdoms: seven or nine? BioSystems 14,461-481. Christen H.R . (1958): Farblose Euglenalen aus dem Hypolimnion des Hausersees. Schweiz. Z. Hydrol. 20, 141-176. Christen H.R. (1959): New colorless Euglenineae.]. Protozool. 6, 292-303. Dawson N.S. and Walne P.L. (1991):Structural characterization of Eutreptia pertyi (Euglenophyta). I. General description. Phycologia 30, 287-302. Dentler WL., Pratt M.M. and Stephens R.E. (1980): Microtubule-membrane interactions in cilia. II Photochemical cross-linking of bridge structures and the identification of a membrane associated dynein -like ATPase.]. Cell BioI. 84, 381-403 . Dragos N., Peterfi L.S. and Popescu C. (1997): Comparative fine structure of pellicular cytoskeleton in Euglena Ehrenberg. Arch. Protistenkd. 148, 277-285. Dubreuil R.R., Marrs J.A. and Bouck G.B. (1992):The cytoskeleton in euglenoids; cell form, surface motility, and cell replication are based on a membrane skeleton of repeating strips. In: Menzel D. (ed.): The cytoskeleton of the algae, pp 59-78. CRC Press, Boca Raton. Gallo ].M. and Schrevel J. (1982j:'Euglenoid movement in Distigmaproteus. I. Cortical rotational motion. BioI. Cell 44,139-143. Hofmann C. and Bouck G.B. (1976): Immunological and structural evidence for patterned intussusceptive surface growth in a unicellular organism. J. Cell BioI. 69, 693-715. Hollande A. (1937): Quelques donnees nouvelles sur la cytologie d'une Astasiacee peu connue : Distigma proteus Ehrenberg. Bull. Soc. zool. France 62, 236-241. Hollande A. (1952): Classe des Eugleniens. In: Grasse P.P. (ed.): Traite de Zoologie, Vol. 1, fasc.1, pp. 238-284. Mason et Ci<, Paris. Kirk ].T.O. and Juniper B.E. (1964):The fine structure of the pellicle of Euglena gracilis. ]. Royal Microscop. Soc. 82,205-210. Lackey J.B. (1934):Studies on the life histories of Euglenida IV. A comparison of the structure and division of Distigma proteus Ehrenberg and Astasiadangeardii Lernrn., a
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