Cortical morphogenesis during encystment in a ciliate, Euplotes encysticus Yonezawa, 1985

European Journal of

Europ.]. Protisto!' 24, 133-137 (1989)

PROTISTOLOGY

Cortical Morphogenesis During Encystment in a Ciliate, Euplotes encysticus Yonezawa, 1985 Tadao Matsusaka and Osamo Noguchi Department of Biology, Faculty of Science, Kumamoto University, Kumamoto, Japan

Fumiaki Yonezawa Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima, Japan

SUMMARY Cortical morphogenesis occurring during encystment was described on the silver impregnated specimens of Euplotes encysticus. During encystment, the ventral cortex of the cell invaginated into the cytoplasm together with the ventral ciliatures at 3 areas, resulting in a reduction of ventral surface. The invagination of the ventral cortex resulted in 3 gaps in the argyrome, which served as the indicators of ventral surface of the cysts. Dorsal bristle rows did not invaginate but remained on the cell surface of the cyst, except for DR 1 which invaginated into the cytoplasm together with AZM, and served as indicators of the dorsal surface of the cyst. Dorso-ventral swellings, which have been observed during encystment of oxytrichid ciliates, did not occur. These findings together with the results of earlier papers suggest that hypotrich cysts should be classified into at least 3 types, 1) Oxytricha-type, 2) Urostyla-type, and 3) Euplotes-type, and that Euplotidae may occupy a phylogenetically distant position to other traditional hypotrichs.

Introduction There have been many papers describing morphogenesis during encystment in the hypotrich ciliates at both light and electron microscope levels. Most species described in these papers, however, are restricted to those belonging to the Oxytrichidae. Diophrys scutum is the only species of Euplotidae, whose encystment has been described ultrastructurally [22], while light microscope descriptions on the process have been made in 3 Euplotes species [6, 8, 26]. In the resting cysts of all these 4 euplotid species, the presence of ciliary organelles have been reported [6, 8, 22, 26], and this is the major difference from the resting cysts of the Oxytrichidae, in which complete absence of ciliary organelles has been demonstrated [10, 11, 16, 17, 21]. Although differences in the resting cyst morphology between Oxytrichidae and Euplotidae might reflect their phylogenetic difference, detailed description on the corti© 1989 by Gustav FischerVerlag,Stuttgart

cal morphogenesis occurring during encystment has not been presented in the species of Euplotidae. The present paper describes the cortical morphogenesis occurring during encystment of Euplotes encysticus Yonezawa, 1985 using silver impregnated specimens.

Material and Methods The hypotrich ciliate, Euplotes encysticus, was grown at 25°C in a dry lettuce extract, to which Bacillus subtilis was inoculated as food organism the day before the transfer of the ciliates. Encystment was induced by simply putting a cell suspension on a cover slip or by the hanging drop method [25]. For the observation of cortical morphogenesis, ciliates were fixed in Champy's fixative and were processed for silver impregnation [4]. The nomenclature of the ciliary organelles is according to Borror [2]. 0932-4739/89/0024-0133$3.50/0

134 . T. Matsusaka, O. Noguchi and F. Yonezawa Results The stationary phase cell was a dorso-ventrally flattened ellipsoid with convex dorsal surface. It measured 64.9 ± 3.5 f-tm in length (mean ± S. D. of 31 Bouin-fixed specimens) and 42.4 ± 2.8 urn in width. The ciliate contained a horseshoe-shaped macronucleus and spherical micronucleus. The following ciliary organelles were observed on the ventral surface: 9 Ironto-ventral cirri (FC l-FC 9), 5 transverse cirri (TC l-TC 5), 2 caudal cirri (CCs), 2 left marginal cirri (LMCs), lapel region of adoral zone of membranelle (AZM), a short paroral membrane (PM), and 2 dorsal bristle rows (DR 1, DR 7) (Fig. la). On the dorsal surface,S longitudinal ridges were observed and at the left side of each ridge, 5 dorsal bristle rows (DR 2-DR 6) were situated (Fig. l b). The collar region of AZM was also observed on the anterior end of the dorsal surface (Fig. 1b). Between the ciliary organelles described above, argyromes were observed on both dorsal and ventral surfaces (Figs. la, b). The first sign of encystment was a translocation of the collar region of the AZM, which moved from the anterodorsal side in the log phase or stationary phase organisms to the antero-ventral region. Fronto-ventral cirri (FCs) began to move toward the anterior. FC 1 to FC 5 arrived close to the right margin of the collar region of the AZM, now located on the antero-ventral surface (Figs. 2a, 3). By this phase the cell shape became nearly round from the dorsal (or ventral) view with a convex dorsal surface but a still flattened ventral surface. The cortex of the adoral area began to be depressed into the cytoplasm together with the AZM and the PM. Following this, the AZM and PM began to invaginate into the cytoplasm. FC 1 to FC 5, now located very close to the collar region of the AZM, also began to invaginate into the depression of the adoral area (Figs. 2b, 4). FC 6 to FC 8 also invaginated into the same depression after the invagination of FC 1 to Fe 5. The

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Fig. 1. Camera lucida drawings of stationary phase cells, showing the disposition of ciliary organelles of ventral (a) and dorsal (b) surfaces. AZM = adoral zone of membranelles, PM = paroral membrane, FC = fronto-ventral cirri, TC = transverse cirri, CC = caudal cirri, LMC = left marginal cirri, DR = dorsal bristle row. Numbers in Figs. show the numbers of FCs and TCs (a) and of DRs (b). Dorsal rows illustrated by dotted lineindicate their locations on dorsal surface (a) or behind dorsal ridge (b). Scale bar = 50 urn,

depression expanded in the left latero-posterior direction. At this point, FC 9 also invaginated into the depression. The cell shape now became almost spherical and the cyst wall began to form. A crescent-shaped gap in the argyrome marked where the invagination of AZM and FCs took place (Figs. 2c, 5). The invagination of TCs began to occur at nearly the same time as the completion of the invagination of the AZM and FCs (Fig. 2c). In this case also, cell cortex surrounding the TCs was depressed and then invaginated. After the invagination, a gap in the argyrome was again observed (Fig. 2d). LMCs and CCs then invaginated into the cytoplasm in the same manner, also resulting in a gap in the argyrome (Figs. 2d, 7). As a result, 3 gaps in the argyrome, which resulted from the invaginations of 1)

Fig. 2. Camera lucida drawings of cortical morphogenesis in 5 successive phases of encystment. a) First phase of encystment. Note the ventral position of the collar region of the AZM and anteriorly disposed FCs. b) Nearly rounded cell showing disappearanceof FC 3 and 4. c) Disappearance of AZM, PM, and FCs leaving a gap in the argyrome (asterisk). Arrow indicates invaginating DR 1. Arrowheads indicate invaginating TCs. d) A cell showing the absence of AZM, PM, FCs, TCs, CCs and LMCs caused by their invagination leaving 3 gaps in the argyrome (asterisks). e) A mature cyst showing the presence of gaps in the argyromes (asterisks). f) The same specimen as (e) showingconvergence of DRs on 2 poles. Scale bar = 50 urn,

Encystment of Euplotes . 135

AZM, PM, FCs, and DR 1 (see below), 2) TCs, and 3) LMCs and CCs, were observed in the ventral cell cortex (Fig. 2d). The gaps became smaller and less conspicuous but persisted even in the mature cysts (Figs. 2e, 8). DRs remained intact on the cell surface of the cyst except for DR 1, which invaginated into the same depression as the AZM at nearly the same time as the TC invagination (Figs. 2c, 6). Thus, 6 DRs were observed on the cell surface of mature cysts. These 6 DRs converged anteriorly and posteriorly, forming 2 poles (Fig. 2f). The gaps in the argyrome and the 2 poles of the convergent DRs are the morphological indicators of the dorso-ventral and anteroposterior axes, respectively. Persistence of dorso-ventral and antero-posterior axes has also been suggested in the cyst of Oxytricha fallax [12, 13], in which no morphological marker of the cell axis is obvious.

The mature cyst was almost spherical with a somewhat flattened ventral surface, which was identified by the gaps in the argyrome. It was 31.6 ± 2.8 urn in diameter (mean ± S.D. of 31 Bouin-fixed specimens) and had Slongitudinal crests similar to those described earlier [3, 6, 18, 26] on its cyst wall, a number that corresponded to the number of dorsal ridges of the vegetative cells. This fact may suggest that dorsal ridges of vegetative cellsplay some role in the crest formation. The orientation of the crests was usually oblique to that of dorsal ridges of the cysts as judged by DRs on the cyst cells (Fig. 9). This may be caused by rotation of encysting cells within newly formed cyst walls. When encystment was induced by placing a drop of cell suspension on a cover slip or by making a hanging drop under a cover slip, most cysts had attached to the cover slips by their ventral surfaces.

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Figs. 3-9. Photomicrographs of silver-impregnated specimens of various phases of encystment. - Fig. 3. A cell in an initial phase of encystment similar to that drawn in Fig. 2a. Note the ventral position of collar region of the AZM, X 800. - Fig. 4. A cell at a similar phase of encystment to that drawn in Fig. 2b. Note the round shape of the cell and invaginating FCs, x 800. - Fig. 5. A cell at a similar phase of encystment to that drawn in Fig. 2c. Arrow indicates the gap in the argyrome, x 800. - Fig. 6. A higher magnification of Fig. 5, showing invaginating DR 1 (thick arrow) and a gap in the argyrome (thin arrow), x 2000. - Fig. 7. A cell in late phase of encystment similar to that drawn in Fig. 2d, showing the gaps in the argyromes (arrows). The gap produced by TC invagination is not in focus, x 800. - Fig. 8. A mature cyst showing gaps in the argyrome (arrows), x 1200. - Fig. 9. Two mature cysts showing the different orientation of crests (short arrows) and DRs (long arrows), x 800.

136 . T. Matsusaka, O. Noguchi and F. Yonezawa Discussion The cyst of Diophrys scutum was the first hypotrich cyst in which a 2-layered cyst wall with a granular layer and intact ciliary organelles was confirmed electron microscopically [22]. Based on these facts, hypotrich cysts have been divided into two groups: 1) kinetosome-resorbing cysts (KR-cysts), which are enclosed within a 3-layered cyst wall and a granular layer and contain no microtubular organelles; and 2) non-kinetosome-resorbing cysts (NKR-cysts), which are enclosed within a 2-layered cyst wall and a granular layer and contain cytoplasmic microtubules, basal bodies and ciliary shafts [22]. At the time the paper appeared, most resting cysts reported electron microscopically were KR-cysts of the Oxytrichidae [9, 10, 14, 15, 23, 24] while the cyst of D. scutum was the only NKR-cyst. The presence of intact ciliary organelles in the resting cyst of E. encysticus was also confirmed electron microscopically (unpublished observation), suggesting the cyst as being a NKR-cyst. Ultrastructural data of the resting cyst are now accumulating and intermediate types between KR- and NKR-cysts have been reported. For example, the resting cysts of Kahliella simplex [7] and Paraurostyla weissei [5] have been reported to have the typical ultrastructural characteristics of a KR-cyst except for the presence of cortical microtubules. A two-layered cyst wall with a granular layer has been reported in the cysts of Urostyla grandis, which contain intact cytoplasmic microtubules and basal bodies but not ciliary shafts [19]. The resting cysts demonstrating Urostyla-type ultrastructures have been observed in our laboratory in Holosticha adami, Pseudourostyla levis and Gonostomum affine (unpublished results). These data suggest that hypotrich cysts should be classified into at least 3 types: 1) the Oxytricha-type cyst, which is enclosed by a 3-layered cyst wall and a granular layer and contains no microtubular organelles, with some exceptions containing only cytoplasmic microtubules; 2) Urostyla-type cyst, which is enclosed by a 2-layered cyst wall and a granular layer and contains cortical mierotubules and basal bodies but not ciliary shafts; and 3) Euplotestype cyst, which is enclosed by a 2-layered cyst wall and a granular layer and contains intact cytoplasmic microtubules, basal bodies, and ciliary shafts. The present and earlier data concerning encystment morphogenesis and resting cyst ultrastructures may suggest the importance of encystment morphogenesis in considerations of ciliate phylogeny. Dorso-ventral swelling and antero-posterior contraction have been reported to occur during encystment of Oxytricha fallax [9], of Pleurotricha sp. [14], and of Histriculus cavicola, erroneously cited as H. muscorum ([15], see also [1]), all belonging to Oxytrichidae. Similar dorso-ventral swelling and antero-posterior contraction were observed during encystment of Holosticha adami, and in another Holosticha species belonging to Urostylidae (unpublished data). In E. encysticus, however, such dorso-ventral swelling did not occur during encystment. Instead, invagination of the ventral cortex together with existing ciliary organelles occurred. These data suggest that the hypotrich ciliates

other than Euplotidae might be phylogenetically closely related, since these ciliates have common encystment morphogenesis. The Euplotidae, on the contrary, might have a different phylogeny from other hypotrichs, since they show different encystment morphogenesis and resting cyst morphology. This consideration seems to be in agreement with a new classification of the phylum Ciliophora [20], in which the Euplotidae were transferred from traditional hypotrich group to another class. Intensive studies to test this are now in progress in our laboratory.

Acknowledgements The authors would like to express their sincere thanks to Dr. Denis H. Lynn (Department of Zoology, University of Guelph, Canada) for his critical reading of the manuscript.

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Key words: Encystment - Euplotes - Silver impregnation - Hypotrich - Phylogeny Tadao Matsusaka, Department of Biology, Faculty of Science, Kumamoto University, Kumamoto 860, Japan