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Reconsideration of the morphogenesis in the marine hypotrichous ciliate, Aspidisca leptaspis Fresenius, 1865 (Protozoa, Ciliophora)
Europ. J. Protistol. 39, 53–61 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ejp
Reconsideration of the morphogenesis in the marine hypotrichous ciliate, Aspidisca leptaspis Fresenius, 1865 (Protozoa, Ciliophora) Weibo Song Laboratory of Protozoology, Ocean University of China, Qingdao 266003, P. R. China; E-mail: [email protected] Received: 4 September 2002; 20 November 2002.
Accepted: 18 January 2003
Morphogenetic events during division of the marine hypotrichous ciliate, Aspidisca leptaspis were investigated. The oral primordium of the opisthe develops hypoapokinetally in a sub-surface pouch posterior to the rear parental adoral zone, as in congeners. The parental adoral zones of membranelles are inherited by the proter. The proter’s buccal cirrus develops from an “extra” anlage, which is formed de novo on the cell surface at an early stage of morphogenesis. Basal bodies in the oral primordium seem not to contribute to the formation of the buccal cirrus of the opisthe, which originates independently within the pouch opposite to the oral primordium and then migrates onto the cell surface. The paroral membrane derives from the posterior end of the oral primordium. Five frontoventral-transverse cirral anlagen are also formed de novo, initially as primary primordia, and develop into 3:3:2:2:2 cirri, respectively, in both dividers. Each of the four dorsal kineties evolves from an intrakinetal anlage in both proter and opisthe. Replication bands progress from the centre to the two ends of the macronucleus during the first half of the morphogenetic process, before this nucleus shortens and divides. The present investigation has clarified division processes within this species and confirmed some morphogenetic differences within the genus. Key words: Morphogenesis; Ciliophora; Hypotrichida; Aspidisca leptaspis.
Introduction Morphogenetic variations, sometimes minor ones, are commonly present among congeners, and can be used to separate species and to analyse the relationships between them (Summers 1935; Borror 1979; Hill 1979; Hemberger 1985; Voss 1989; Wang et al. 1992; Eigner and Foissner 1992; Foissner 1996). In some groups, such as the euplotids, very similar or even identical patterns of morphogenesis are found in all congeners within such genera as Uronychia, Euplotes and Diophrys (Wallengren 1900; Shimomura 1967; Washburn and Borror 1972; Tuffrau et al. 1976; Hill 1979, 1981a,b; Voss 1989; Song and Packroff 1993; Wang and Song 1995; Foissner 1996; Shi and Song 1998). Evidence about morphogenetic
events during binary fission of Aspidisca is rather fragmentary, but a number of descriptions suggest that these events vary between species in this genus (Chatton 1942; Bonner 1954; Wise 1965; Diller 1966; Ruffolo 1976; Czapik 1981; Matsusaka et al. 1989; Fleury 1991; Wang et al. 1997). The living morphology and infraciliature of the large marine species Aspidisca leptaspis Fresenius, 1865 were redescribed by Song and Wilbert (1997). The morphogenesis of the same species was investigated by Wang et al. (1997) under the name A. pulcherrima Kahl, 1935. This account (in Chinese) is not generally accessible, and, since some stages were overlooked or misinterpreted, the protargol0932-4739/03/39/01-053 $ 15.00/0
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impregnated slides have been re-examined to prepare the more detailed description presented here. Comparison with descriptions of morphogenesis in other Aspidisca species confirms that morphogenetic patterns are not uniform in this genus.
Material and methods Samples from several populations of Aspidisca leptaspis were collected repeatedly between 1992 and 1995 from coastal waters near Qingdao (36°08′ N; 120°43′ E), China. Cells were isolated and cultured in the laboratory using methods described before (Song and Wilbert 1994). Specimens undergoing morphogenesis were examined in protargol impregnated slides, prepared by the method of Wilbert (1975). Drawings were made with the help of a camera lucida at 1250× magnification. For clarity, parental cirri are shown in diagrams of morphogenetic stages only by outline (except Fig. 1A), whereas new ones are shaded black. Terminology is mainly according to Kahl (1932) and Curds and Wu (1983).
Results Morphology and identification of Aspidisca leptaspis (Figs 1A, B; 3A) Recently Aspidisca leptaspis Fresenius, 1865 was morphologically redescribed and redefined by
Song and Wilbert (1997). As observed by the present and previous authors (Kahl 1932; Wu and Curds 1979; Song and Wilbert 1997), its morphology and infraciliature have revealed extremely high stability, except for the posterior spines, which appear to vary from insignificant to conspicuous. Based on the present observations and descriptions obtained on populations/strains all over the world, this species is characterized by 1) oval body shape with 2 conspicuous left-lateral and several smaller posterior spines; 2) seven large (including one “buccal cirrus”) and one small frontoventral (FV) cirri, of which the latter is posteriorly adjacent to the rightmost large FV cirrus; 3) five transverse cirri, of which the leftmost one is slender and often divided in vivo into 2–3 subunits; 4) long adoral zone part 2 (AZM2) consisting of ca. 20 membranelles; 5) seven to eight small membranelles in AZM1 which is positioned typically in the leftfrontal area and 6) 4 densely ciliated dorsal kineties, which are arranged along the inconspicuous dorsal ridges.
Morphogenesis in cell division of Aspidisca leptaspis (Figs 1–4) The earliest cortical morphogenetic event is the appearance of a small patch of basal bodies (kinetosomes) in irregular arrangement, the oral primordium (OP), which occurs de novo beneath the
Fig. 1. Early and middle division stages of Aspidisca leptaspis from protargol-impregnated specimens. A, B. Ventral and dorsal side of the same specimen, to show the newly-formed frontoventral-transverse anlagen (arrows in A), the oral primordium (double arrowheads) and the macronuclear replication bands (arrows in B); arrowhead in A marks the buccal cirrus. C. Macronucleus, with the replication bands (arrows). D. Ventral side, to show that 5 cirral anlagen appear as primary primordia on the cell surface, while the oral primordium is developing beneath the parental AZM2 (arrow). Note that the anlage for the buccal cirrus appears de novo anterior to the oral primordium (double arrowheads). E. Ventral side, to demonstrate that the buccal cirrus of the opisthe appears near the oral primordium (arrow), while the double arrowheads indicate the buccal cirrus of the proter. Note that the primary cirral primordia divide into two groups (CA1, CA2). F, G. Ventral and dorsal side of the same specimen, double arrowheads in F mark the anterior part of the newly-formed membranelles in the oral primordium, which is about to separate from its posterior portion; arrow in F indicates the buccal cirrus of the opisthe. Note that the cirral anlagen begin to segregate. The replication of the macronucleus is almost competed (arrows in G). H, I. Ventral and dorsal view of the same specimen, to show that anlagen for dorsal kineties are newly formed within the old structures (arrows in I), while all new cirri are formed. Note that the oral primordium is still beneath the pellicle (and the old AZM). Arrow in H marks the anterior end of the oral primordium, double arrowheads indicate the buccal cirrus. J, K. Ventral and dorsal view of the same specimen. Arrow in J marks the newly-separated anterior part of the adoral membranelles (AZM1), which is still under the old AZM2; double arrowheads indicate the paroral membrane for the opisthe, while arrows in K refer the kinetosomes proliferated within the old dorsal kineties. Note that the macronucleus is deformed and just preparing to divide. AZM1, AZM2 = first (anterior) and second (posterior) part of adoral zone of membranelles; CA1, CA2 = first (anterior) and second (posterior) group of cirral anlagen; CP = cirral primordia; CyP = cytopharynx; DK = dorsal kineties; FVC = frontoventral-transverse cirri; Ma = macronucleus; PM = paroral membrane; TC = transverse cirri. Scale bars: 40 µm.
Morphogenesis of Aspidisca leptaspis
cortex of the ventral surface, conspicuously posterior to the AZM2 and to the left of the transverse cirri (Fig. 1A, double arrowheads). After initial OP formation, there is rapid proliferation of basal bodies with development of a subsurface pouch, in which growth of the OP continues (Fig. 1D, arrow). By this time, a small patch of basal bodies has appeared near the AZM2 as a new anlage of the buccal cirrus for the proter (Fig. 1D, double arrowheads).
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At about the same time as the OP appears, basal bodies for cirral anlagen (CP) develop as 3 fine and short streaks anterior to the transverse cirri on the cell surface. No parental ciliatures are involved in the formation of these new streaks (Fig. 1A, arrows). Slightly later, two other cirral anlagen are formed de novo to the right of the parental rightmost FVcirrus. In the end of this stage, each of 5 cirral anlagen extends to its maximum length (Fig. 1D, CP).
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Fig. 2. Late division stages of Aspidisca leptaspis from protargol-impregnated specimens. A, B. Ventral and dorsal view of the same specimen, to show that the first part of the adoral zone of membranelles (AZM1, arrow) begins to migrate anteriorly; double arrowheads mark the new buccal cirrus in the proter; note that all cirri are completely differentiated; arrows in B mark the presumptive division furrow of dividers. C. Ventral side, arrow marks the anterior lateral spine, double arrowheads indicate the small cirrus deriving from the anlage 5. Note the AZM1 is moving onto the cell surface. D, E. Ventral and dorsal view of the same individual just before division, arrows in D mark the old cirri that still remain; arrow in E indicates that the macronucleus is about to complete division. F, G. Daughter cells just after division (F, proter; G, opisthe), arrows in both figures mark the reduced old cirri, while double arrowheads demonstrate the macronucleus just separated. AZM1, = first (anterior) part of adoral zone of membranelles; I–V = first to fifth cirral anlage; Ma = macronucleus; PM = paroral membrane. Scale bars: 40 µm.
Fig. 3. Photomicrographs of Aspidisca leptaspis in morphogenesis. A. Ventral view, arrow marks the small frontoventral cirrus. B. Cell in vivo (slightly deformed !), note the spines on left cell margin. C, D. Early stages of dividers, arrows in C mark the thread-like FVT-anlagen. E. Middle division stage, arrow indicates the oral primordium. F. Ventral view, to show the fragmented cirri. G. Ventral view of a late divider, arrow marks the newly-segregated AZM1. H. To show the small frontoventral cirrus. I. The same individual as H, to demonstrate the deformed macronucleus. J. Ventral view of a late divider, arrow marks the paroral membrane in the opisthe, while double arrowheads refer the small frontoventral cirrus.
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Fig. 4. Photomicrographs of Aspidisca leptaspis in morphogenesis. A. Ventral view, to demonstrate anterior part of the adoral zone of membranelles (AZM1) in proter (black arrow) and in the opisthe (white arrow). B. To show the macronucleus just before division (arrow). C, D. Very late dividers, arrow in C marks AZM1 of the opisthe. E, F. To demonstrate the macronucleus during division.
The ciliature on the dorsal surface is unchanged at this time. At the centre of the macronucleus two replication bands are recognizable (Fig. 1B, arrows), these subsequently move along the arms si-
multaneously to both ends of the macronucleus (Fig. 1C, G, arrows). Division continues with the formation of cirral anlagen fields of the proter and opisthe. These
Morphogenesis of Aspidisca leptaspis
streaks then slightly broaden and become completely separated into anterior and posterior groups (as CA1, and CA2 in Fig. 1E). As the pouch of the OP enlarges, the basal bodies align into membranelles from the leftmost end and organize towards the right (Fig. 1F, double arrowheads). A new independent anlage for the buccal cirrus of the opisthe is formed, which seems to be on the ventral wall of the subcortical pouch, while clearly not derived from the OP (Fig. 1E, arrow). In the next stage the two sets of cirral anlagen enlarge and begin to break apart and develop as distinct cirri. The buccal cirrus develops in both the proter and the opisthe (Fig. 1E, double arrowheads; Fig. 1F, single arrow). The resorption of some parental cirri is about to begin (Fig. 1F), while the ciliature on the dorsal side remains unchanged (Fig. 1G). In the following period, the anterior end (leftmost end) of the opisthe’s adoral zone of membranelles emerges onto the cortical surface (Fig. 1H, arrow). The posterior part, as well as the buccal cirrus, is still within the subsurface pouch (Fig. 1H, double arrowheads); both of these will soon completely migrate outside. At this morphogenetic stage the formation of all adoral membranelles in the opisthe is still in process, while the segregation of new frontoventral-transverse (FVT) cirri (including the buccal cirrus) from the FVT-anlagen is nearly finished (Fig. 1H). At about the same time, the proliferation of new basal bodies occurs separately in both anterior and posterior portions of each parental dorsal kinety. Later these will develop into 2 groups of kinetal rows (anlagen) for both dividers (Fig. 1I, arrows). The migration of replication bands in the micronucleus is about completed by this stage. Figures 1J and K show a mid-late divider. Conspicuous events in this period are the separation of the two cirral sets from each other and the formation of the paroral membrane in the opisthe, which is clearly derived from the posterior-most part of the oral primordium (Fig. 1J, double arrowheads). The formation of the membranelles of the opisthe in the OP is almost completed and the final number of membranelles can now be recognized. The AZM2 moves onto the cell surface following elongation of the cell. The anterior part (the leftmost portion, the developing AZM1 of the opisthe), which has separated from the main part by migrating anteriorly, seems to be still lying within the subsurface pouch (Fig. 1J, arrow). The buccal
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cirrus of the opisthe moves anteriorly on the cell surface to the level of the other frontoventral cirri (see also Fig. 1H, double arrowheads). On the dorsal side, the proliferation of new basal bodies occurs at two levels in the dorsal kineties, which are then rapidly elongated (Fig. 1K, arrows). The macronucleus has shortened and thickened into a compact sausage-shaped mass. As division proceeds, all cirri migrate to their final positions. In both proter and opisthe the five FVT-cirral anlagen are seen to have given rise to 3:3:2:2:2 mature frontoventral and transverse cirri, respectively. The buccal cirrus in both dividers has already reached the final size. The AZM1 in the opisthe is still beneath the cell surface (Fig. 2A, arrow). Along with the formation of the division furrow, development and migration of all cirri and other ciliary organelles are completed (Fig. 2C, D). Also finished is the division of the macronucleus (Fig. 2E, arrow). The resorption of parental cirri is well advanced (Fig. 2D, arrows). Each of the dorsal kineties extends to the ends of both cells. Figures 2F and G show the proter (F) and opisthe (G) just after division. Morphogenetic events of all cortical structures have been completed except that the body shape, size and general position of some ciliary organelles are still not exactly the same as in the trophic cell. The resorption of parental cirri is almost completed (Fig. 2F, G, arrows). The macronucleus will soon re-extend to the shape and position of their form in interphase. In summary, all parental cirri are replaced by new structures during morphogenesis. The proter retains both parts of the parental membranelles, with new membranelles of both AZM1 and AZM2 being developed in the opisthe. In both proter and opisthe a buccal cirrus originates from an anlage formed de novo. Additional cilia are developed intrakinetally within the dorsal kineties of both proter and opisthe.
Discussion The characteristics of A. leptaspis have been reexamined. Considering the variability of the spines on the cell margin, the body shape and size, the habitat, the number of frontoventral cirri and especially the general appearance of AZM1 and AZM2, (size and number of membranelles), several morphotypes were synonymized with Aspidisca leptaspis by Song and Wilbert (1997): A. sedigita
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Quennerstedt, 1867, A. hexeris Quennerstedt, 1869, A. crenata Fabre-Domergue, 1885, A. pulcherrima Kahl, 1932, A. sedigita sensu Dragesco, 1963, A. tridentata Dragesco, 1963, A. pulcherrima sensu Tuffrau, 1964, A. baltica sensu Borror, 1968, A. caspica Agamaliev, 1967, A. psammobiotica Burkovsky, 1970 and A. lyncaster sensu Fleury et al., 1986. The population described by Tuffrau (1964) under the name Aspidisca lyncaster (Müller 1786) Stein, 1859, we believe, in agreement with Wu and Curds (1979), to have actually been a population of A. leptaspis, and we have regarded his account as being of the latter species in the present paper. The morphogenesis of seven nominal species of Aspidisca has been described previously: A. cicada (Brown 1966), A. costata (Hamm 1964; Diller 1975; Hill 1979; Pang and Fu 1992), A. orthopogon (Deroux and Tuffrau 1965), A. aculeata (Dini and Bacchi 1976; Wang et al. 1992), A. lynceus (Summers 1935), A. lyncaster (Tuffrau 1964) and A. pulcherrima (Wang et al. 1997). Slightly different events occur during binary fission of these congeners, particularly with respect to the formation of some frontoventral cirri. Thus, according to the origin and numbering (following Wallengren, 1900) of the FVT cirri, at least three different types are known: the leptaspis-type, the orthopogon-type and the costata-type, in which the cirral anlagen IV give rise to patterns of 3:3:2:2:2 (Tuffrau 1964, Wang et al. 1997), 3:3:2:3:1 (totally 7 frontoventral and 5 transverse cirri formed) (Deroux and Tuffrau 1965) and 3:3:2:2:1 (6 frontoventral and 5 transverse cirri) (Hill 1979; Pang and Fu 1992), respectively. Thus, Aspidisca with 8 “frontoventral” cirri (including one buccal cirrus) exhibit the first two types, while those with 7 cirri (also including one buccal cirrus) possess the last type of the pattern. The morphogenesis of A. leptaspis was first described by Tuffrau (1964) from a Mediterranean strain, identified by him as A. lyncaster, then redescribed by Wang et al. (1997), based on a Qingdao population identified by them as A. pulcherrima. These two descriptions left uncertainties about the origins of the buccal cirrus and paroral membrane in the opisthe, and about how the cirral anlagen segregated. In addition, Tuffrau (1964) interpreted incorrectly that the anterior part of the AZM (the AZM1) in the opisthe originates from an anlage that is separate from that of the AZM2 (as he depicted in his Fig. 5e, page 182). In fact this separate anlage seen by Tuffrau represents the newlyformed primordium of the buccal cirrus, and the
primordia of the two parts of the AZM only separate from one another later. These uncertainties have been resolved in the present study. Song and Wilbert (1997) doubted the validity of Aspidisca orthopogon Deroux & Tuffrau, 1965 when they described the morphology of A. leptaspis. These species can be distinguished, however, by both morphology (the number of membranelles and the position of the small frontoventral cirrus) and morphogenetical characters. The anlagen I-–V form 3:3:2:3:1 cirri, respectively, during morphogenesis in A. orthopogon vs. 3:3:2:2:2 in A. leptaspis, although both species possess 8 frontoventral cirri (including one “buccal cirrus”). Aspidisca orthopogon should thus be recognised as a clearly defined species. The origin and mode of formation of some frontoventral-transverse cirri and the AZM1 in Aspidisca described by Diller (1975) differed from that described here. According to his interpretation, the fragmentation of FVT cirri in the costata-type (with 7 frontoventral cirri including one “buccal cirrus”) followed a pattern of 3:3:2:2:2 rather than 3:3:2:2:1 (i.e. one more cirrus is formed), while the AZM1 was described as originating from the “buccal cirrus-anlage” (called anlage I in his account). Compared with data from related morphotypes, especially those possessing the same cirral pattern, that is A. costata and A. aculeata (Pang and Fu 1992; Wang et al. 1992), we believe that these conclusions of Diller (1975) were erroneous. Without exception, the AZM1 is derived from the anterior part of the oral primordium in all congeners so far investigated ontogenetically. Acknowledgements: This work was supported by “the National Science Foundation of China” (Project No. 30170114) and the “Chueng Kong Scholars Programme”. I am grateful to Mr. Gong Jun, graduate student in the Laboratory of Protozoology, OUC, for his technical assistance for computer treatment of some illustrations.
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Morphogenesis of Aspidisca leptaspis Chatton E. (1942): Le problème de la continuité du cinétome chez les hypotriches. Nouvelles recherches sur l’ Euplotes crassus. Bull. Biol. Fr. Belg. 76, 314–335. Curds C.R. and Wu I.C.H. (1983): A review of the Euplotidae (Hypotrichida, Ciliophora). Bull. Br. Mus. nat. Hist. (Zool.) 44, 191–247. Czapik A. (1981): La morphogenèse chez le cilié Diophrys oligothrix Borror. Acta Protozool. 20, 367–372. Deroux G. and Tuffrau M. (1965): Aspidisca orthopogon n. sp. Révision de certains mécanismes de la morphogenèse a l’aide d’une modification de la technique au protargol. Cah. Biol. Mar. 6, 293–310. Diller W.F. (1966): Correlation of ciliary and nuclear development in the life cycle of Euplotes. J. Protozool. 13, 43–54. Diller W.F. (1975): Nuclear behavior and morphogenetic changes in fission and conjugation of Aspidisca costata (Dujardin). J. Protozool. 22, 221–229. Dini F. and Bacchi P. (1976): Ciclo cellulare di Aspidisca aculeata (Ehrenberg). Atti Acad. Nat. Dei Lincei, Cl. Sci. Fis. Mat. Nat., ser. 8, 64–69. Eigner P. and Foissner W. (1992): Divisional morphogenesis in Bakuella pampinaria nov. spec. and reevaluation of the classification of the urostylids (Ciliophora, Hypotrichida). Europ. J. Protistol. 28, 460–470. Fleury A. (1991): Dynamics of the cytoskeleton during morphogenesis in the ciliate Euplotes I. Basal bodies related microtubular system. Europ. J. Protistol. 27, 99–114. Foissner W. (1996): Ontogenesis in ciliated protozoa, with emphasis on stomatogenesis. In Hausmann K. & Bradbury P. C. (eds.) Ciliates: cells as organisms. Gustav Fischer, Stuttgart. pp. 95–177. Fresenius G. (1865): Die Infusorien des Seewasseraquariums. Zool. Gart. Frankf. 6, 81–89, 121–129. Hamm A. (1964): Untersuchungen über die Ökologie und Variabilität von Aspidisca costata (Hypotrichida) in Belebtschlamm. Arch. Hydrobiol. 60, 283–339. Hemberger H. (1985): Neue Gattungen und Arten hypotricher Ciliaten. Arch. Protistenkd. 130, 397–417. Hill B.F. (1979): Reconsideration of cortical morphogenesis during cell division in Aspidisca (Ciliophora, Hypotrichida). Trans. Am. Microsc. Soc. 98, 537–542. Hill B.F. (1981a): The cortical morphogenesis cycle associated with cell division in Diophrys Dujardin, 1841 (Ciliophora, Hypotrichida). J. Protozool. 28, 215–221. Hill B.F. (1981b): Uronychia transfuga (O. F. Müller, 1786) Stein, 1859 (Ciliophora, Hypotrichia, Uronychiidae): cortical structure and morphogenesis during division. J. Protozool. 37, 99–107. Kahl A. (1932): Urtiere oder Protozoa I: Wimpertiere oder Ciliata (Infusoria). Tierw. Deutschl. 25, 399–650. Matsusaka T., Noguchi O. and Yonezawa F. (1989): Cortical morphogenesis during encystment in a ciliate, Euplotes encycticus Yonezawa, 1985. Europ. J. Protistol. 24, 133–137. Pang Y. and Fu Z. (1992): The morphology and morphogenesis on Aspidisca costata. J. East China Normal. Univ. (Suppl.) pp 91–100 (in Chinese with English summary). Ruffolo J.J. (1976): Cortical morphogenesis during the cell division cycle in Euplotes: an integrated study using light optical, scanning electron and transmission electron microscopy. J. Morph. 148, 489–527.
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Shi X. and Song W. (1998): On morphology and morphogenesis of Uronychia setigera during asexual division. Acta Hydrobiol. Sin. 23, 146–151 (in Chinese with English summary). Shimomura T. (1967): Cell division and stomatogenesis in Euplotes. Mie Medical J. 17, 93–107. Song W. (1995): Morphogenetic studies on Uronychia uncinata (Protozoa, Ciliophora) during its asexual division. Acta Oceanol. Sin. 15, 93–99. Song W. and Packroff G. (1993): Beitrag zur Morphogenese des marinen Ciliaten Diophrys scutum (Dujardin, 1841). Zool. Jb. Anat. 123, 85–95. Song W. and Wilbert N. (1994): Morphogenesis of the marine ciliate Diophrys oligothrix Borror, 1965 during the cell division (Protozoa, Ciliophora, Hypotrichida). Europ. J. Protistol. 30, 38–44. Song W. and Wilbert N. (1997): Morphological investigations on some free living ciliates (Protozoa, Ciliophora) from China Sea with description of a new hypotrichous genus, Hemigastrostyla nov. gen. Arch. Protistenkd. 148, 413–444. Summers F.M. (1935): The division and reorganization of Aspidisca lynceus Müller, Diophrys appendiculata Stein, and Stylonychia pustulata Ehrbg. Arch. Protistenkd. 85, 173–208. Tuffrau M. (1964): La morphogenèse de bipartition et les structures neuromotrices dans le genre Aspidisca (ciliés hypotriches). Revue de quelques espèces. Cah. Biol. Mar. 5, 173–199. Tuffrau M., Tuffrau H. and Genermont J. (1976): La réorganisation infraciliaire au cours de la conjugaison et l’origine du primordium buccal dans le genre Euplotes. J. Protozool. 23, 517–523. Voss H.J. (1989): Vergleichende morphogenetische Untersuchung von 13 Arten der Gattung Euplotes (Ciliophora, Hypotrichida). Arch. Protistenkd. 137, 331–344. Wallengren J. (1900): Zur kenntnis der vergleichenden Morphologie der hypotrichen Infusorien. Bih. K. svenska Vetensk. Akad. Handl. 26, 1–31. Wang C., Zhou Y. and Pang Y. (1992): Studies on morphology and morphogenesis of Aspidisca aculeata. J. East China Norm. Univ. (Spec. Iss. Neurobiol. Protozool.) 106–114 (in Chinese with English summary). Wang M., Mu Z., Zhang S. and Song W. (1997): Morphogenesis of Aspidisca pulcherrima (Protozoa: Ciliophora). Zool. Res. 18, 185–188 (in Chinese with English summary). Wang M. and Song W. (1995): Morphogenetic studies on the marine ciliate Euplotes charon (Protozoa, Ciliophora). Zool. Res. 16, 233–238 (in Chinese with English summary). Washburn E.S. and Borror A.C. (1972): Euplotes raikovi Agamaliev, 1966 (Ciliophora, Hypotrichida) from New Hampshire: description and morphogenesis. J. Protozool. 19, 604–608. Wilbert N. (1975): Eine verbesserte Technik der Protargolimprägnation für Ciliaten. Mikrokosmos 64, 171–179. Wise B.N. (1965): The morphogenetic cycle in Euplotes eurystomus and its bearing on problems of ciliate morphogenesis. J. Protozool. 12, 626–648. Wu I.C.H. and Curds C.R. (1979): A guide to the species of the genus Aspidisca. Bull. Br. Mus. Nat. Hist. (Zool.) 36, 1–34.
Reconsideration of the morphogenesis in the marine hypotrichous ciliate, Aspidisca leptaspis Fresenius, 1865 (Protozoa, Ciliophora) Weibo Song Laboratory of Protozoology, Ocean University of China, Qingdao 266003, P. R. China; E-mail: [email protected] Received: 4 September 2002; 20 November 2002.
Accepted: 18 January 2003
Morphogenetic events during division of the marine hypotrichous ciliate, Aspidisca leptaspis were investigated. The oral primordium of the opisthe develops hypoapokinetally in a sub-surface pouch posterior to the rear parental adoral zone, as in congeners. The parental adoral zones of membranelles are inherited by the proter. The proter’s buccal cirrus develops from an “extra” anlage, which is formed de novo on the cell surface at an early stage of morphogenesis. Basal bodies in the oral primordium seem not to contribute to the formation of the buccal cirrus of the opisthe, which originates independently within the pouch opposite to the oral primordium and then migrates onto the cell surface. The paroral membrane derives from the posterior end of the oral primordium. Five frontoventral-transverse cirral anlagen are also formed de novo, initially as primary primordia, and develop into 3:3:2:2:2 cirri, respectively, in both dividers. Each of the four dorsal kineties evolves from an intrakinetal anlage in both proter and opisthe. Replication bands progress from the centre to the two ends of the macronucleus during the first half of the morphogenetic process, before this nucleus shortens and divides. The present investigation has clarified division processes within this species and confirmed some morphogenetic differences within the genus. Key words: Morphogenesis; Ciliophora; Hypotrichida; Aspidisca leptaspis.
Introduction Morphogenetic variations, sometimes minor ones, are commonly present among congeners, and can be used to separate species and to analyse the relationships between them (Summers 1935; Borror 1979; Hill 1979; Hemberger 1985; Voss 1989; Wang et al. 1992; Eigner and Foissner 1992; Foissner 1996). In some groups, such as the euplotids, very similar or even identical patterns of morphogenesis are found in all congeners within such genera as Uronychia, Euplotes and Diophrys (Wallengren 1900; Shimomura 1967; Washburn and Borror 1972; Tuffrau et al. 1976; Hill 1979, 1981a,b; Voss 1989; Song and Packroff 1993; Wang and Song 1995; Foissner 1996; Shi and Song 1998). Evidence about morphogenetic
events during binary fission of Aspidisca is rather fragmentary, but a number of descriptions suggest that these events vary between species in this genus (Chatton 1942; Bonner 1954; Wise 1965; Diller 1966; Ruffolo 1976; Czapik 1981; Matsusaka et al. 1989; Fleury 1991; Wang et al. 1997). The living morphology and infraciliature of the large marine species Aspidisca leptaspis Fresenius, 1865 were redescribed by Song and Wilbert (1997). The morphogenesis of the same species was investigated by Wang et al. (1997) under the name A. pulcherrima Kahl, 1935. This account (in Chinese) is not generally accessible, and, since some stages were overlooked or misinterpreted, the protargol0932-4739/03/39/01-053 $ 15.00/0
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impregnated slides have been re-examined to prepare the more detailed description presented here. Comparison with descriptions of morphogenesis in other Aspidisca species confirms that morphogenetic patterns are not uniform in this genus.
Material and methods Samples from several populations of Aspidisca leptaspis were collected repeatedly between 1992 and 1995 from coastal waters near Qingdao (36°08′ N; 120°43′ E), China. Cells were isolated and cultured in the laboratory using methods described before (Song and Wilbert 1994). Specimens undergoing morphogenesis were examined in protargol impregnated slides, prepared by the method of Wilbert (1975). Drawings were made with the help of a camera lucida at 1250× magnification. For clarity, parental cirri are shown in diagrams of morphogenetic stages only by outline (except Fig. 1A), whereas new ones are shaded black. Terminology is mainly according to Kahl (1932) and Curds and Wu (1983).
Results Morphology and identification of Aspidisca leptaspis (Figs 1A, B; 3A) Recently Aspidisca leptaspis Fresenius, 1865 was morphologically redescribed and redefined by
Song and Wilbert (1997). As observed by the present and previous authors (Kahl 1932; Wu and Curds 1979; Song and Wilbert 1997), its morphology and infraciliature have revealed extremely high stability, except for the posterior spines, which appear to vary from insignificant to conspicuous. Based on the present observations and descriptions obtained on populations/strains all over the world, this species is characterized by 1) oval body shape with 2 conspicuous left-lateral and several smaller posterior spines; 2) seven large (including one “buccal cirrus”) and one small frontoventral (FV) cirri, of which the latter is posteriorly adjacent to the rightmost large FV cirrus; 3) five transverse cirri, of which the leftmost one is slender and often divided in vivo into 2–3 subunits; 4) long adoral zone part 2 (AZM2) consisting of ca. 20 membranelles; 5) seven to eight small membranelles in AZM1 which is positioned typically in the leftfrontal area and 6) 4 densely ciliated dorsal kineties, which are arranged along the inconspicuous dorsal ridges.
Morphogenesis in cell division of Aspidisca leptaspis (Figs 1–4) The earliest cortical morphogenetic event is the appearance of a small patch of basal bodies (kinetosomes) in irregular arrangement, the oral primordium (OP), which occurs de novo beneath the
Fig. 1. Early and middle division stages of Aspidisca leptaspis from protargol-impregnated specimens. A, B. Ventral and dorsal side of the same specimen, to show the newly-formed frontoventral-transverse anlagen (arrows in A), the oral primordium (double arrowheads) and the macronuclear replication bands (arrows in B); arrowhead in A marks the buccal cirrus. C. Macronucleus, with the replication bands (arrows). D. Ventral side, to show that 5 cirral anlagen appear as primary primordia on the cell surface, while the oral primordium is developing beneath the parental AZM2 (arrow). Note that the anlage for the buccal cirrus appears de novo anterior to the oral primordium (double arrowheads). E. Ventral side, to demonstrate that the buccal cirrus of the opisthe appears near the oral primordium (arrow), while the double arrowheads indicate the buccal cirrus of the proter. Note that the primary cirral primordia divide into two groups (CA1, CA2). F, G. Ventral and dorsal side of the same specimen, double arrowheads in F mark the anterior part of the newly-formed membranelles in the oral primordium, which is about to separate from its posterior portion; arrow in F indicates the buccal cirrus of the opisthe. Note that the cirral anlagen begin to segregate. The replication of the macronucleus is almost competed (arrows in G). H, I. Ventral and dorsal view of the same specimen, to show that anlagen for dorsal kineties are newly formed within the old structures (arrows in I), while all new cirri are formed. Note that the oral primordium is still beneath the pellicle (and the old AZM). Arrow in H marks the anterior end of the oral primordium, double arrowheads indicate the buccal cirrus. J, K. Ventral and dorsal view of the same specimen. Arrow in J marks the newly-separated anterior part of the adoral membranelles (AZM1), which is still under the old AZM2; double arrowheads indicate the paroral membrane for the opisthe, while arrows in K refer the kinetosomes proliferated within the old dorsal kineties. Note that the macronucleus is deformed and just preparing to divide. AZM1, AZM2 = first (anterior) and second (posterior) part of adoral zone of membranelles; CA1, CA2 = first (anterior) and second (posterior) group of cirral anlagen; CP = cirral primordia; CyP = cytopharynx; DK = dorsal kineties; FVC = frontoventral-transverse cirri; Ma = macronucleus; PM = paroral membrane; TC = transverse cirri. Scale bars: 40 µm.
Morphogenesis of Aspidisca leptaspis
cortex of the ventral surface, conspicuously posterior to the AZM2 and to the left of the transverse cirri (Fig. 1A, double arrowheads). After initial OP formation, there is rapid proliferation of basal bodies with development of a subsurface pouch, in which growth of the OP continues (Fig. 1D, arrow). By this time, a small patch of basal bodies has appeared near the AZM2 as a new anlage of the buccal cirrus for the proter (Fig. 1D, double arrowheads).
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At about the same time as the OP appears, basal bodies for cirral anlagen (CP) develop as 3 fine and short streaks anterior to the transverse cirri on the cell surface. No parental ciliatures are involved in the formation of these new streaks (Fig. 1A, arrows). Slightly later, two other cirral anlagen are formed de novo to the right of the parental rightmost FVcirrus. In the end of this stage, each of 5 cirral anlagen extends to its maximum length (Fig. 1D, CP).
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Fig. 2. Late division stages of Aspidisca leptaspis from protargol-impregnated specimens. A, B. Ventral and dorsal view of the same specimen, to show that the first part of the adoral zone of membranelles (AZM1, arrow) begins to migrate anteriorly; double arrowheads mark the new buccal cirrus in the proter; note that all cirri are completely differentiated; arrows in B mark the presumptive division furrow of dividers. C. Ventral side, arrow marks the anterior lateral spine, double arrowheads indicate the small cirrus deriving from the anlage 5. Note the AZM1 is moving onto the cell surface. D, E. Ventral and dorsal view of the same individual just before division, arrows in D mark the old cirri that still remain; arrow in E indicates that the macronucleus is about to complete division. F, G. Daughter cells just after division (F, proter; G, opisthe), arrows in both figures mark the reduced old cirri, while double arrowheads demonstrate the macronucleus just separated. AZM1, = first (anterior) part of adoral zone of membranelles; I–V = first to fifth cirral anlage; Ma = macronucleus; PM = paroral membrane. Scale bars: 40 µm.
Fig. 3. Photomicrographs of Aspidisca leptaspis in morphogenesis. A. Ventral view, arrow marks the small frontoventral cirrus. B. Cell in vivo (slightly deformed !), note the spines on left cell margin. C, D. Early stages of dividers, arrows in C mark the thread-like FVT-anlagen. E. Middle division stage, arrow indicates the oral primordium. F. Ventral view, to show the fragmented cirri. G. Ventral view of a late divider, arrow marks the newly-segregated AZM1. H. To show the small frontoventral cirrus. I. The same individual as H, to demonstrate the deformed macronucleus. J. Ventral view of a late divider, arrow marks the paroral membrane in the opisthe, while double arrowheads refer the small frontoventral cirrus.
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Fig. 4. Photomicrographs of Aspidisca leptaspis in morphogenesis. A. Ventral view, to demonstrate anterior part of the adoral zone of membranelles (AZM1) in proter (black arrow) and in the opisthe (white arrow). B. To show the macronucleus just before division (arrow). C, D. Very late dividers, arrow in C marks AZM1 of the opisthe. E, F. To demonstrate the macronucleus during division.
The ciliature on the dorsal surface is unchanged at this time. At the centre of the macronucleus two replication bands are recognizable (Fig. 1B, arrows), these subsequently move along the arms si-
multaneously to both ends of the macronucleus (Fig. 1C, G, arrows). Division continues with the formation of cirral anlagen fields of the proter and opisthe. These
Morphogenesis of Aspidisca leptaspis
streaks then slightly broaden and become completely separated into anterior and posterior groups (as CA1, and CA2 in Fig. 1E). As the pouch of the OP enlarges, the basal bodies align into membranelles from the leftmost end and organize towards the right (Fig. 1F, double arrowheads). A new independent anlage for the buccal cirrus of the opisthe is formed, which seems to be on the ventral wall of the subcortical pouch, while clearly not derived from the OP (Fig. 1E, arrow). In the next stage the two sets of cirral anlagen enlarge and begin to break apart and develop as distinct cirri. The buccal cirrus develops in both the proter and the opisthe (Fig. 1E, double arrowheads; Fig. 1F, single arrow). The resorption of some parental cirri is about to begin (Fig. 1F), while the ciliature on the dorsal side remains unchanged (Fig. 1G). In the following period, the anterior end (leftmost end) of the opisthe’s adoral zone of membranelles emerges onto the cortical surface (Fig. 1H, arrow). The posterior part, as well as the buccal cirrus, is still within the subsurface pouch (Fig. 1H, double arrowheads); both of these will soon completely migrate outside. At this morphogenetic stage the formation of all adoral membranelles in the opisthe is still in process, while the segregation of new frontoventral-transverse (FVT) cirri (including the buccal cirrus) from the FVT-anlagen is nearly finished (Fig. 1H). At about the same time, the proliferation of new basal bodies occurs separately in both anterior and posterior portions of each parental dorsal kinety. Later these will develop into 2 groups of kinetal rows (anlagen) for both dividers (Fig. 1I, arrows). The migration of replication bands in the micronucleus is about completed by this stage. Figures 1J and K show a mid-late divider. Conspicuous events in this period are the separation of the two cirral sets from each other and the formation of the paroral membrane in the opisthe, which is clearly derived from the posterior-most part of the oral primordium (Fig. 1J, double arrowheads). The formation of the membranelles of the opisthe in the OP is almost completed and the final number of membranelles can now be recognized. The AZM2 moves onto the cell surface following elongation of the cell. The anterior part (the leftmost portion, the developing AZM1 of the opisthe), which has separated from the main part by migrating anteriorly, seems to be still lying within the subsurface pouch (Fig. 1J, arrow). The buccal
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cirrus of the opisthe moves anteriorly on the cell surface to the level of the other frontoventral cirri (see also Fig. 1H, double arrowheads). On the dorsal side, the proliferation of new basal bodies occurs at two levels in the dorsal kineties, which are then rapidly elongated (Fig. 1K, arrows). The macronucleus has shortened and thickened into a compact sausage-shaped mass. As division proceeds, all cirri migrate to their final positions. In both proter and opisthe the five FVT-cirral anlagen are seen to have given rise to 3:3:2:2:2 mature frontoventral and transverse cirri, respectively. The buccal cirrus in both dividers has already reached the final size. The AZM1 in the opisthe is still beneath the cell surface (Fig. 2A, arrow). Along with the formation of the division furrow, development and migration of all cirri and other ciliary organelles are completed (Fig. 2C, D). Also finished is the division of the macronucleus (Fig. 2E, arrow). The resorption of parental cirri is well advanced (Fig. 2D, arrows). Each of the dorsal kineties extends to the ends of both cells. Figures 2F and G show the proter (F) and opisthe (G) just after division. Morphogenetic events of all cortical structures have been completed except that the body shape, size and general position of some ciliary organelles are still not exactly the same as in the trophic cell. The resorption of parental cirri is almost completed (Fig. 2F, G, arrows). The macronucleus will soon re-extend to the shape and position of their form in interphase. In summary, all parental cirri are replaced by new structures during morphogenesis. The proter retains both parts of the parental membranelles, with new membranelles of both AZM1 and AZM2 being developed in the opisthe. In both proter and opisthe a buccal cirrus originates from an anlage formed de novo. Additional cilia are developed intrakinetally within the dorsal kineties of both proter and opisthe.
Discussion The characteristics of A. leptaspis have been reexamined. Considering the variability of the spines on the cell margin, the body shape and size, the habitat, the number of frontoventral cirri and especially the general appearance of AZM1 and AZM2, (size and number of membranelles), several morphotypes were synonymized with Aspidisca leptaspis by Song and Wilbert (1997): A. sedigita
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Quennerstedt, 1867, A. hexeris Quennerstedt, 1869, A. crenata Fabre-Domergue, 1885, A. pulcherrima Kahl, 1932, A. sedigita sensu Dragesco, 1963, A. tridentata Dragesco, 1963, A. pulcherrima sensu Tuffrau, 1964, A. baltica sensu Borror, 1968, A. caspica Agamaliev, 1967, A. psammobiotica Burkovsky, 1970 and A. lyncaster sensu Fleury et al., 1986. The population described by Tuffrau (1964) under the name Aspidisca lyncaster (Müller 1786) Stein, 1859, we believe, in agreement with Wu and Curds (1979), to have actually been a population of A. leptaspis, and we have regarded his account as being of the latter species in the present paper. The morphogenesis of seven nominal species of Aspidisca has been described previously: A. cicada (Brown 1966), A. costata (Hamm 1964; Diller 1975; Hill 1979; Pang and Fu 1992), A. orthopogon (Deroux and Tuffrau 1965), A. aculeata (Dini and Bacchi 1976; Wang et al. 1992), A. lynceus (Summers 1935), A. lyncaster (Tuffrau 1964) and A. pulcherrima (Wang et al. 1997). Slightly different events occur during binary fission of these congeners, particularly with respect to the formation of some frontoventral cirri. Thus, according to the origin and numbering (following Wallengren, 1900) of the FVT cirri, at least three different types are known: the leptaspis-type, the orthopogon-type and the costata-type, in which the cirral anlagen IV give rise to patterns of 3:3:2:2:2 (Tuffrau 1964, Wang et al. 1997), 3:3:2:3:1 (totally 7 frontoventral and 5 transverse cirri formed) (Deroux and Tuffrau 1965) and 3:3:2:2:1 (6 frontoventral and 5 transverse cirri) (Hill 1979; Pang and Fu 1992), respectively. Thus, Aspidisca with 8 “frontoventral” cirri (including one buccal cirrus) exhibit the first two types, while those with 7 cirri (also including one buccal cirrus) possess the last type of the pattern. The morphogenesis of A. leptaspis was first described by Tuffrau (1964) from a Mediterranean strain, identified by him as A. lyncaster, then redescribed by Wang et al. (1997), based on a Qingdao population identified by them as A. pulcherrima. These two descriptions left uncertainties about the origins of the buccal cirrus and paroral membrane in the opisthe, and about how the cirral anlagen segregated. In addition, Tuffrau (1964) interpreted incorrectly that the anterior part of the AZM (the AZM1) in the opisthe originates from an anlage that is separate from that of the AZM2 (as he depicted in his Fig. 5e, page 182). In fact this separate anlage seen by Tuffrau represents the newlyformed primordium of the buccal cirrus, and the
primordia of the two parts of the AZM only separate from one another later. These uncertainties have been resolved in the present study. Song and Wilbert (1997) doubted the validity of Aspidisca orthopogon Deroux & Tuffrau, 1965 when they described the morphology of A. leptaspis. These species can be distinguished, however, by both morphology (the number of membranelles and the position of the small frontoventral cirrus) and morphogenetical characters. The anlagen I-–V form 3:3:2:3:1 cirri, respectively, during morphogenesis in A. orthopogon vs. 3:3:2:2:2 in A. leptaspis, although both species possess 8 frontoventral cirri (including one “buccal cirrus”). Aspidisca orthopogon should thus be recognised as a clearly defined species. The origin and mode of formation of some frontoventral-transverse cirri and the AZM1 in Aspidisca described by Diller (1975) differed from that described here. According to his interpretation, the fragmentation of FVT cirri in the costata-type (with 7 frontoventral cirri including one “buccal cirrus”) followed a pattern of 3:3:2:2:2 rather than 3:3:2:2:1 (i.e. one more cirrus is formed), while the AZM1 was described as originating from the “buccal cirrus-anlage” (called anlage I in his account). Compared with data from related morphotypes, especially those possessing the same cirral pattern, that is A. costata and A. aculeata (Pang and Fu 1992; Wang et al. 1992), we believe that these conclusions of Diller (1975) were erroneous. Without exception, the AZM1 is derived from the anterior part of the oral primordium in all congeners so far investigated ontogenetically. Acknowledgements: This work was supported by “the National Science Foundation of China” (Project No. 30170114) and the “Chueng Kong Scholars Programme”. I am grateful to Mr. Gong Jun, graduate student in the Laboratory of Protozoology, OUC, for his technical assistance for computer treatment of some illustrations.
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