The cyst of urostyla grandis (hypotrichida : Urostylidae) : ultrastructure and evolutionary implications

PROTISTOLOGICA , 1985, T. XXI, fase. 4, p. 481-485 . (HYPOTRICHIDA THE CYST OF UROSTYLA GRANDIS UROSTYLIDAE) : ULTRASTRUCTURE AND EVOLUTIONARY IMPLICATIONS R.M. RIOS, A. TORRES, P. CALVO and C. FEDRIAN I Departamento de Microbiología, Facultad de Biología , Universidad de Sevilla, Sevilla, España ABSTRACT U ltrastructural study of Urostyla grandis cysts shows that the cyst wall is constitued of three layers :a fibrous ectocyst, a structurally homogeneous endocyst and a granular layer that exhibits a dispersed granular or fibrillar structure. The maintenance of only a few kinetosomes and scattered bundles of subpellicular microtubules, and other particular ities distinguish the Urostyla grandis cyst, both KR and NKR cysts. It is proposed to amplify the WALKER and MAUGEL classification by an additional group. Based on comparative features, sorne of the phylogenetic relations are discussed. Key words : cyst, ultrastructure, hypotrich, Urostyla. classification. RÉSUM É L'examen , en microscopie électronique, du kyste d' Uros1y la grandis a révélé que sa paroi se compose de trois couches : un exokyste fibrillaire, un endokyste a structure homogéne et une couche granulaire avec une structure granulaire ou fibrillaire. Le maintien de quelques cinétosomes, microtubules subpellicula ires et d'autres particularités permettent de distinguer ces kystes des kystes KR et NKR. On propose d'ajouter un nouveau groupe a la classification de WALKER et MAUG EL. On commente quelques relations phylogéniques reposan! sur des caractéristiques comparatives. Mots-c:lés : kyste, ultrastructure, hypotriche, Urostyla. classification. INTRODUCTION Only a few species of the Oxytrichidae have been examined since 1980, (CALVO et al.. 1983; GuTJERREZ et al., 1983; MATSUSAKA and HONGO, 1984; VERNI et al.. 1984), According to the ultrastructural characteristics displayed by the cysts of hypotrich ciliates, WALKER and MAUGEL in 1980, proposed a general classification for these of two groups : KR cysts (kinetosome-resorbing cysts) : Oxytricha fallax (GRJM ES, 1973), Gastrostyla steinii (WALKER et al.. 1980) and Stylonichia mytilus (WALKER et al., 1975). NKR cysts (no kinetosome-resorbing cysts) : Dyophris scutum (WALKER and MAUGEL, 1980). but ali of them exhibit KR cysts; so apparently, KR cysts may be a feature of the Oxytrichidae. In the same way, WALK ER and MA UGEL (1980) suggest that the N KR cysts seem to be a characteristic of the Euplotidae. In this paper, we describe the ultrastructural topography and fine structure of the cyst of the hypotrich ciliate Urostyla grandis. Special emphasis has been placed on the fact that the Urostyla grandis cyst displays characteristics between KR and N KR cysts and we suggest the Manuscrit recu le 21 mai 1985; accepté par le Comité de lecture le 19 septembre 1985. 481 possibility of the establishment of an additional group, including species showing similar resting cysts. These ultrastructural variations may be a new criterion offered to taxonomists to resolve both taxonomic and phylogenetic problems in the Order Hypotrichida. MATERIALS AND METHODS Urostyla grandis. a hypotrich ciliate, was cultivated at 20 ± 1 °C in mineral water and fed Ch/orogonium sp. In cultu res of Urostyla grandis. encystment occurs when food becomes a limiting factor. In order to induce this process, to obtain resting cysts, vegetative cells were transferred from the maintenance culture to mineral water without adding C/orogonium sp. Staining for light mlcroscopy The nuclear apparatus was stained by using the Feulgen procedure (TORRES, 1977). Electron microscopy For scanning electron microscopy (SEM), the cysts were fixed for 30 minutes at 4 °C in cacodylate buffer O.OS M pH 7.2, containing a 1 :2 mixture of 1.S o/o glutaraldehyde and 2 o/o osmium tetroxide. The fixed material was dehydrated by increasing concentrations of acetone, was air-dried, then gold-coated and finally examined in a Jeol scanning electron microscope. For transmission electron microscopy (TEM), the cysts were fixed for 40 minutes at 4 °C in a mixture 1 : 2 of l .S o/o glutaraldehyde and 2 % osmium tetroxide in a O.OS M cacodylate buffer pH = 7.2. The fixed material was embedded in 2 o/o agar blocks and dehydrated by increasing concentrations of acetone and embedded in Spurr resin (SPURR, 1968). Ultrathin sections were cut with a Reichert-J ung ultramicrotome, double stained with uranyl-acetate and lead citrate and then examined with a Siemens Elmiscop 102 electron microscope. Alternatively, the cysts were stained with ruthenium red (Lu FT, 1971) and then processed like those mentioned above. RESULTS Light mlcroscopy The Urostyla grandis cysts are spherical and average 22-24 µm in diameter. The volume of the cyst is smaller than that of the vegetative cell. The cytoplasm exhibits an homogeneous aspect and in older cysts it is possible to observe a dark granule, in the center, that stands out clearly from the rest of the cytoplasm (Fig. 1). Cysts possess the normal vegetative nuclear com plement consisting of a lot of macronuclei and a few micronuclei (Fig. 2). Electron microscopy In electron micrograph s obtained by SEM, the cyst surface appears irregular with wrinkles that give a rough F1G. 1. - Photomicrograph of a resting cyst of Urosty/a gra11dis "in vivo". e, cytoplasm ; g, central granule; w, cyst wall. (1400 x ). F1G. 2. - Resting cyst of Urosty/a gra11dis showi ng a lot of macronuclear fragments. Stained by Feulgen . {1400 x ). F1G. 3. - Scanni ng electron micrograph of a resting cyst of Urosryla gra11dis. ( 1400 x ). F1G. 4. - Transmission electron micrograph of a cyst showing cyst wall, remainding subpellicular microtubules and mucocysts. ec. ectocyst ; en, endocyst ; gl, granular layer: mt. subpellicu lar microtubules; mu, mucocyst. {27 000 x ). F1G. 5. - Transmission electron micrograph of the cyst wall stai ned with ruthenium red. ec, ectocyst; en, endocyst; gl, gran ular layer: ( 1. - outer ectocyst - 2. - inner ectocyst). (33 000 x ). F1G. 6. - Transmission electron micrograph. Cyst wall and cyst pellicle . ec, ectocyst; en, endocyst ; gl, granu lar !ayer: p, pellicle. (45 000 x ). F1G . 7. - Transmission electron micrograph showing a pair of kinetosomes beneath thc cyst wall. k, ki netosome s. (30 000 x ). F1G. 8. - Mitochondrial cluster. ( 16 000 x ). F1G. 9. - Cyst cytoplasm showi ng single mitochondria closely associated to RER and Golgi apparatus. Note t rans1t1on vesicles. mi, mitochondria; G, Golgi apparatus; tv, transition vesicles; R, rough endoplasmic ret iculum. (50 000 x ). 482 483 aspee!. Ostioles or pores have never been observed on the wrinkled mature cyst surface; although, it is possible to see sorne bacteria adhering to the folds (Fig. 3). Observations of ultrathin sections reveal that the cyst wall of Urostyla grandis consists of three morphologi cally distinct layers (Figs. 4, 6 and 7). The outer most layer (ectocyst), the thickest one, is composed of fibrillar material which has a low electron density. For this reason, the structure of this layer is very difficult to observe, but in cysts stained with ruthenium red, two zones can be clearly recognized : the outer zone formed by fibrous material randomly oriented and less packed and th e inner zone, also fibrillar and more packed (Fig. 5). The endocyst is very homogeneous and regular both in shape and thickness. lt is made u p of an electro-dense, highly compact, amorphou s substance. The granular layer, the most internal one, appears fibrillar in sorne sections and granular in others. The thickness of this layer is not uniform since it is uniting the spherical cyst wall to the grooved cell surface. As a consequence of the loss of the external membrane-unit, the cyst pellicle, beneath the cyst wall , presents one membrane-unit less than that of the vegeta tive cell ( Fig. 6). Likewise, the network of subpellicular microtubules, although visible in young cysts, disappears al most completely. In the mature cysts, it is possible to observe sorne remai ns at only a few point s underneath the pellicle (Fig. 4). The function of these remainders of subpellicular microtubules in the cyst is unknown, but we think they might ensure the maintenance of the shape during the encystment. The only cili atu re we could observe consists of a few isolated pairs of ki netosomes (Fig. 7). Mitochondria can be seen to be formed in tight clusters or scattered throughout the cytoplasm (Fig. 8). When they appear isolated a closely association with rough endoplasmic reticulum and Golgi apparatus has been noted and , in every case, the transition vesicles are clearly observed (Fig. 9). Both the vegetative cell and resti ng cyst present numerous mucocysts, bu! in the vegetative cell those organelles exhibit several different aspects, apparently representing different stages of development , while in the resting cyst ali of them show the same appearance. They appear as highly osmiophilic-granule s surrounded by a double membrane (Fig. 4). Unlike the vegetative cell, no fusion of mucocysts with the pellicle was noted in the resting cysl. DISCUSSION Our observations obtai ned by TEM of Urostyla grandis cyst show that the wall consists of three layers, like the Dyophris scutum cyst (WALK ER and MA UGEL, 1980). On the other hand, the morphology of these layers is basically similar to that of the three i nner layers of Oxytrichid s cysts (mesocyst, endocyst and granular layer). According to this 484 result, we could say that the Urostyla grandis cyst seems to lack the ectocyst present in all Oxytrichids cysts described. No data have been reported indicating structural differences between the cyst pellicle and the vegetative cell pellicle. However, in Urostyla grandis cyst, the outer most membran e-unit, which always appears in the vegetative state, has never been observed . lt may be due to the degradation of this structure during the encystment process; although another possibility also exists. This membrane-unit can remain intact but is masked by the endocyst. Studies in progress , in our laboratory, seem to indicate that the second option is the most probable one and that it is between this membrane-unit and the rest of the pellicle where the material that will form the granular layer will be left. This layer shows a variety of appearan - ces, but, in every case, the cytoplasm matches it. The significance of this fact is as yet unknown . In most of the ultrathin sections a few remains of ciliature, consisting of one, two or several kinetosomes can be observed . This characteristic distinguishes the Urostyla grandis cyst from the KR cyst, where the ciliature disappears almost completely, and the N KR cyst, in which the ciliature stays almost intact (incl uding AZM). Another singu lar characteristic of these cysts is the fact that the subpellicular microtubu les are reduced to severa( scattered bundles beneath the pellicle . Since the numerous macronuclear fragments observed in the vegetat ive cell can be found i n the cyst, it is possible to conclude that macronuclear fusion <loes not take place during the encystment process. No appreciable changes are noticeable in the macronuclear chromatin arrangement; although, occasionally , a small increase of the condensation degree could be seen. In agreement with the classification proposed by WALKER and MA UGEL ( 1980) (KR and NKR cysts), ali the species belonging to the Oxytrichidae family, whose cysts have been studied, exhibit KR cysts. In addition, WA LK ER and M A UGEL ( 1980) suggest that the NKR cysts may be a feature of the Eu plotidae. However, this classification looses its validi ty in the Urostyla grandis cysts because they show mixed characteristics of both types. Sorne observations of other species of Urostylids reveal that their cysts exhibit the same characteristics described in this paper (unpublished data). For these reason s, we think it is necessary to amplify the classification with a third grou p, which includes ali the species whose cysts : (1) exhibit a three-layered wall, (2) resorb ali cilia but maintain a few kinetosomes and microtubules, (3) display mitochondrial clusters, (4) do not fuse macronuclei, (5) rate cyst volume/vegetative cell volume = 0.25-0.40 and (6) rate cyst wall thickness/cyst radius = 0.08 ± 0.02. The encystment can be considered as a process of cell differentiation to defend itself against possible environmental challenges. Two general types of hypotrich s cysts (KR and N KR) exist as an adjustment for survival in different environmental conditions. As suggested by different authors, the main adaptative advantages of the formation of N KR cysts seem to be their rapid ability of excystment and their lower energy required for this process, as a consequence of the conservation of ali thei r cortical structures. With regard to the K R cysts, their higher resistance to extrema! values of the environm ental parameters (due to thicker wall) is an advantage which withstands the loss of valuable cortical markers such as cilia and kinetosomes . In respect to this, the Urostyla grandis cyst appears to be situated half-way between the two, since its resistance properties are lower than the KR cysts, but the ciliary structures that remain i n the resti ng form (kinetosom es and subpellicular microtubule s) may be useful indicators of morphogenetically determina - tive position s for the excystment process. Up to the present time, the formation of the cyst wall i n a few ciliates has been assumed to be the result of mucocysts discharge (CHEISSEN and MOSEVICH, 1962: HOLT and CHAPMAN, 1971 ; Me ARDLE et al., 1980; REPAK and PFISTER, 1967), at least partially. But, in hypotrichs, the appearance of mu cocysts in the cyst has never been reported . In Urostyla grandis (and other species of U rostylidae family, unpublished data) it is possible to observe a lot of these organelles, both in the vegetative cell and the resting form, although their functional role in the encystment process still remains obscure. At this time, in our laboratory, we are studying the structure and function of mucocysts in encysting Urostyla grandis. lf we assume that the cyst described in this report is the general type of the Urostylidae cysts, in our opinion, sufficient data exist to establish a correlation between the morphogenetic division pattern s and the cyst types. So, we suppose that the four-layered cyst present in the Oxytrichidae which exhibits a type of morphogenetical pattern characterizing evolved forms of hypotrich s is derived from the three-layered cyst present in the Urostylids whose morphogen etical pattern characterizes the more primitives hypotrichs. 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