Oxytricha bifaria (ciliata, hypotrichida)

Europ.], Protisto!' 23, 343-349 (1988)

European Journal of

PROTISTOLOGY

Oxytricha bifaria (Ciliata, Hypotrichida) General Morphology and Ultrastructure of Normal Cells and Giants* Giovanna Rosati, Angela Giari and Nicola Ricci Dipartimento di scienze dell' ambiente e del territorio, Universita di Pisa, Italia

SUMMARY Both the general morphology and the ultrastructure distinguish the giants (cannibals) of Oxytri-

chabifaria from the normal cells. Beyond the general change of the body shape (i.e. differential increase of the thickness of the body along its major axis), a quantitative variation of some of the ciliary organelles (in somatic ciliature the increase of the number of the serially repeating structures and of the frontal cirri; in oral ciliature the increase of the number of membranelles and of the number of cilia per membranelle; the increase of the ciliary rows of the external paroral membrane) has been evidenced together with a differential modification of certain cortical areas. It can be concluded that it is quite appropriate to speak of cell differentiation referring to the giants of O. bifaria.

Introduction

Oxytricha bifaria, a fresh water hypotrich ciliate [3] spends about 95% of its active life cycle as an organism growing and dividing vegetatively. However, to face the challenges possibly encountered in a widely variable environment such as that of internal waters, this species acquired the capability to differentiate reversibly into several forms: the pairs [14J, generally ensuring a periodical reassortment of the genetic pool; the cysts [17), representing a sort of life boat when severe prolonged environmental stresses affect the species; and the cannibals [16J that are gigantic forms produced when overcrowding conditions occur. These giants have already been investigated from the point of view of macro- and micro-nuclear DNA content [19] while a study is still in progress regarding their adaptative significance. The general picture of this form, emerging from the above mentioned studies, suggested that it was also worth undertaking a study of its ultrastructure. As an exhaustive picture of O. bifaria at the ultrastructural level has not been available in the literature up to now, we report in this paper a description of both normal cells and giants in order to illustrate the differences .. This work was supported by grants from the Consiglio Nazionale delle Ricerche and Ministero della Pubblica Istruzione. © 1988 by GustavFischerVerlag, Stuttgart

between these two different forms and, possibly, to account for at least some of the giants' peculiarities.

Material and Methods Cells of the stock S6 which easily produce giants under controlled conditions, were used throughout our experiments: the cultures were fed and handled according to the techniques reported elsewhere [15]. For scanning electron microscopy (SEM) the cells were fixed with a 1: 1 mixture of 5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, and 2% OS04 in distilled water: then they were put on poly-L- lisine-coated coverslips, dehydrated in ethanol and, after critical point drying, they were coated with gold and examined with a Philips microscope SEM 505. For transmission electron microscopy (TEM), after fixation as above, the cells were dehydrated in ethanol and embedded in Epon-Araldite mixture. The sections were stained by uranyl acetate followed by lead citrate. Measurements were performed by different methods: 1) The measures of cell dimensions were taken from SEM pictures, except for those concerning thickness, which were obtained on cross semithin sections seen in the light microscope (Table 1). 2) The composition and the dimensions of the somatic ciliature components (Tables 2 and 3) resulted from SEM pictures. 3) The data concerning the oral apparatus (Table 4) were obtained partly from SEM pictures (length of the paroral externe and of the lapel) and partly from TEM pictures (dimensions of the cytopharynx). 0932-4739/88/0023-0343$3.50/0

344 . G. Rosati, A. Giari and N. Ricci Table 1. Thickness of normal and gigantic O. bifaria as measured on cross sections

Table 3. Variation of dimensions of the FC zone Length (urn)

Normal cells (urn) Giants (11m)

18 28

23 40

25 45

23 40

21 36

Absolute increase (11m) % increase

10 55

17 75

20 80

17 75

15 71

Width (urn) Distance fromVC (urn)

Normal cells

Note: 5 specimens of both normal cells and giants were studied for this purpose:the standard deviations are not indicated being very small, normally less than 10% of the relative mean values.

5C SD

26 30 28.5 22.5 26 26.6 2.8

12 11.2 11.5 13.5 13 12.24 0.98

25 23 26.4 26 24 24.8 1.4

5C SD

33 32 35 36 36 34.4 1.8

14.5 13 14 13 14 13.7 0.6

36 36 38 37.5 36 36.7 0.9

29.3

12.6

47.9

Giants 4) The quantitative variations of the proximal AZM (Table 5) were obtained from TEM pictures. Whenever statistically analyzed, the different sets of data were compared by the Student's T test.

% increase

Results

5C = mean; SD = standard deviation; FC = frontal cirri; VC = ventral cirri.

I) General morphology a) Normal cells

Oxytricha bifaria is oval in shape and dorso-ventrally flattened. The cells of the stock used in this work are 80 urn long and 45 urn wide on the average (n = 50). The thickness reaches the maximum (25 urn) in the middle of the body and slopes regularly towards both anterior and posterior ends (Table 1). Thus, a normal O. bifaria can be approximated to a half ellipsoid (Fig. 1).

II) Somatic ciliature a) Normal cells

b) Giants As already reported by Ricci and Riggio [16] the giants are easily recognizable among the other cells at the stereomicroscope level: their cytoplasm is dark, their shape is very irregular and the anterior end appears widened and squared. They are also larger than normal oxytricha reaching 150 urn in length and 80 in width (n = 50). As shown in Table 1 giants are also thicker than the normal cells although the increase in thickness does not follow a regular pattern: in particular, it is lower at the anterior end than at the posterior end. This results in a change of the general shape of the body. In fact, a giant has a shape similar to that of a halved pear (Fig. 3).

Six kineties, for convenience numbered from left to right as seen in the figure [8], are present on the dorsal surface (Fig. 1). Rows 1-3 extend antero-posteriorly forming a curve with the convexity at the left and contain 20-30 bristle units. Row 4, which consists of 26 bristles in most cells observed, is bent in the opposite direction together with rows 5 and 6. The latter two rows are shorter than all the others. The spacing between contiguous dorsal bristles in each row is fairly constant (Table 2). The ventral somatic ciliature (Fig. 2) consists of two major groups of ciliary structures: 1) a series of cirri positioned in the following groups: 8 frontal (FC), 3 ventral (VC), 7 transverse (TC). Three caudal (CC) are generally inserted just at the posterior limit between the ventral and the dorsal surface but, often, they are entirely dorsal. 2) The marginal cirral rows (LMC and RMC) extend from anterior to posterior on the left and right margins of the ventral surface respectively.

Table 2. Differential composition of somatic ciliature Dorsal Normal cells n = 30 Giants n = 20

5C

DR

BR4

BS

RMC

6

26 1.6

2.8 0.3

28.2 2.3

6

34 1.8

2.8 0.3

38.0 2.9

SD

5C SD

Ventral FC

VC

TC

CC

8

3

7

3

10.7 0.7

3

7

3

n = number of cells; 5C = mean; SD = standard deviation; DR = dorsal rows; BR 4 = bristle row 4; BS = bristle spacing; RMC = right marginal cirri; FC = frontal cirri; VC = ventral cirri; CC = caudal cirri.

Ultrastructure of O. bifaria Giants . 345 Table 4. Dimensional variations of the oral apparatus Lapel

Paroral externe (urn)

X = 21 ± 2.3

Normal cells

n = 30

X = 44.6 ± 2.2

Giants

n

P.A.

(urn)

= 28

31 ± 2.3 n = 30 52 ± 2.4 n = 28

14° ± 40° n = 30 30° ± 6° n = 28

Cytopharynx Length

Width

(urn)

(urn)

4.2 ± 0.36 n=5 7.6 ± 0.58 n=5

2.2 ± 0.2 n = 5 2.8 ± 0.5 n=5

x = mean; P.A. = peristomial angle. Table5. Quantitative variations of the proximal AZM Intermembranellar Number of cilia per row row1 row2 row3 row4 spacing (um) Normal cells n= 5 Giants n=5

3

15-16 22-23 22-23 X = 1.3 ± 0.18

3

32

44

44

X = 1.5 ± 0.23

n = number of cells; X = mean.

were counted in all the giants examined (Fig. 4). The "additional" FC are inserted in the naked zones to the left of the two FC which in normal cells lie singly. Thus, in the case of 12 FC, 4 rows of 3 cirri each can be observed in the frontal area. As reported in Table 3 the increase in width and length of the area in which FC are inserted is lower than that measured for the distance between FC and ve. The number of VC, TC and CC remains constant like the length of cilia measured for FC and RMe.

III) Buccal apparatus a) Normal cells

In order to have precise references in comparing normal and giant cells, right marginal and frontal cirri were examined more carefully as representatives of the two major groups of the ventral ciliary structures. The data concerning this analysis are reported in Tables 2 and 3. The number of right marginal cirri shows a certain degree of variation. The number of FC is, on the contrary, definitely constant: 8 cirri were counted in all the normal cells observed, without exceptions. As shown in Fig. 2 they occupy the region at the right of the oral apparatus and are arranged as follows: starting from the anterior end of the cell a first group of 3 cirri is settled in a row; below the left-hand cirrus (namely the most central one of these cirri) lies a single cirrus, a second row of 3 cirri follows and, finally, another single cirrus is inserted posteriorly. The distance between the latter cirrus, that is the posteriormost of FC and the anteriormost of VC was measured in 5 cells. The values are reported in Table 3 together with the data concerning the length and the width of the region in which FC are inserted. The length of the cilia is about 10 lim (SD = 2.2 ) in both RMC and Fe. b) Giants The transformation of normal O. bifaria into giants does not include a variation in the number of dorsal kineties (Fig. 3). However, the number of the bristle units per row increases while the spacing between contiguous bristles (Table 2) and their general organization remain unchanged. As for the ventral ciliature, a significant (P < 0.01) increase in the number of RMC was observed (Table 2). Surprisingly, also the number of FC increases: 10-12 cirri

The peristome is triangular in shape and extends for about 1/3 of the ventral surface. Along the anterior and left margins is a series of membranelles (the adoral zone of membranelles, AZM) , while along the right margin two undulating membranes or parorales are present. They are inserted on different sides of a cytoplasmic ridge: the one on the peristomial side (at the left of the ridge) is called paroral intern (Pi), while the other, inserted at the right, is called paroral extern (Pe). The length of Pe and that of the lapel (i.e. the portion of AZM bordering the left peristomial margin) were chosen as parameters for the comparison between normal and giant oral apparatuses, together with the angle of the peristomial funnel between these same ciliary structures. The data for normal cells are reported in the upper row of Table 4. As revealed by ultrastructural analysis each membranelle consists of 4 parallel rows of kinetosomes (Fig. 5): the first row, the anteriormost, is the shortest one as it consists of only 3 kinetosomes, the second consists of 15-16 kinetosomes and the remaining two of 22-23 (see Table 5). These values concern the portion of AZM just above the opening of the cytostome (proximal region). In the same region the intermembranellar spacing was also measured as the distance from the base of a membranelle and the neighboring free space to an equivalent point of the adjacent membranelle. The Pi consists of a single row of kinetosomes while the Pe consists of two rows (Fig. 8). In Fig. 7 a section of the terminal part of the peristome is seen at TEM. Beneath the level of the posteriormost adoral membranelles the buccal cavity forms the cytopharynx: a sort of channel limited by a single unit membrane. The data concerning the length and the width of this structure are reported in Table 4.

346 . G. Rosati, A. Giari and N. Ricci

Figs. 1-4. SEM micrographs of O. bifaria. - Fig. 1. dorsal view of a normal cell. - Fig. 2. ventral view of a normal cell. - Fig. 3. dorsal view of a giant. - Fig. 4. ventral view of a giant. The dorsal kineties are progressively numbered. FC = frontal cirri; VC = ventral cirri; IC = transversal cirri. Arrows indicate the supernumerary Fe. Scale bars represent 20 urn.

Ultrastructure of O. bifaria Giants . 347

b) Giants

As reported in the lower row of Table 4, the length of both the lapel and the Pe and the angle between these two structures are significantly greater (P < 0.01) than in normal cells. By means of ultrastructural observation also a variation in the number of the cilia per membranelle was evidenced (Fig. 6): except for the shortest row, which does not change, the number of kinetosomes in the other membranellar rows is nearly double in giants (Table 5). In contrast to this, the intermembranellar spacing does not change. Thus, the absolute number of the membranelles, although not exactly determined, cannot but be higher in giants than in normal cells. The Pi consists of one row of kinetosomes as in normal cells while the Pe consists of three rows instead of two (Fig. 9). The cytopharynx (Fig. 10) appears significantly longer (P < 0.01) than in normal cells, showing only a little, not significant variation in width (Table 4). Discussion

I) General morphology From studies carried out on the oxytrichid Paraurostyla [2] it is evident that an adjustment of cortical pattern to cell dimensions takes place including proportional changes not only in the number of the elements but also in their constitution. When a normal O. bifaria becomes a giant, a variation in size takes place in which the proportions between width and length remain nearly the same: however, the giant does not retain the regular ellipsoid shape typical of normal cells (see Table 1) because its anterior part enlarges only slightly compared to the other regions of the body. This may well be due to the absence of ingested food organisms in this region, recalling the "A zone" of S. mytilus [9]: the presence of a cytopharynx in O. bifaria accounts for an "A zone" specifically restricted to the anterior part.

II) Somatic ciliature The number of the structures of the somatic ciliature being serially repeated (namely the dorsal bristle kineties and the marginal cirral rows) is greater in giants than in normal cells. This fact was not unexpected because these structures are known to vary in number very widely even within the same species, their number being rather correlated with the cell size [1]. The relative distance between two of these elements is kept constant in normal cells and giants as already reported for Paraurostyla [10]. As to the ventral ciliature the most relevant change observed in the giants is the increase in number of FC which represent a group of cirri very constant in number not only in O. bifaria but also in all species belonging to the Stylonychia-Oxytricha group [1] although Alonso and Perez da Silva (1963) reported that the number of ventral cirri in giant stylonychias is larger than in normal cells. The modality and the timing of this change remain still unknown. The only evident trait is that they are unfailingly localized in the same region, perfectly settled in rows

with the normal ones. As the area occupied by FC in giants increases only slightly, they appear crowded although well ordered. It is already known that the cell surface in ciliates is not uniformly morphogenetically competent [11]. It is possible that also the functional control of very specialized structures such as cirri is committed to specific competent regions of the cortex which cannot vary their size except within a defined range. In this connection, it is worth mentioning that a hypothesis has been proposed [4, 13,22,23] according to which recent evolutionary events in ciliates are correlated with the oligomerization of ciliary structures. This phenomenon, the direct consequence of which is a grouping of the ciliary structures (usually constant in oxytrichids [24]), cannot but be associated with a differentiation in the functional significance of the different cortical regions. Thus the fact that the increase in dimensions in giants of O. bifaria is higher in the naked zone between FC and VC than in the FC area, was not unexpected. Although indirect, this way of looking at the whole story seems to be supported by another consideration: Blepharisma (Ciliata Heterotrichida) changes its cell size continuously depending on the dimensions of the organisms it feeds on [7, 20] while O. bifaria maintains its own species-specific size when fed on species smaller than 40-50 urn and shifts onto a well adaptive pathway differentiating the gigantic forms when the dimensions of the food organisms are larger than 60-80 urn (unpublished data from Cetera, Banchetti, Ricci). Such a discontinuous and dramatic change in the morphology seems to support what was mentioned above about the specific differentiation and morphogenetic constraints of the hypotrichs in comparison with the less differentiated heterotrichs. The other cirral groups, namely VC, TC, and CC, maintain their number. This differential behavior of apparently similar structures is a further indication that the transformation of normal O. bifaria in giant cells, far from being a simple increase in size, represents a true differentiative process which follows a well-defined and highly constant pattern. III) Buccal apparatus

The whole peristomial area appears widened in giants proportionally to the whole body: most of the variations observed in the oral ciliature follow the rule that therelative proportions of the elements are retained when a variation in size occurs. So, for example, not only the number of membranelles but also the number of kinetosomes in each membranellar row (except for the shortest one) is higher in giants than in normal cells. This finding not only perfectly fits into the general picture drawn by the reports of Fenchel [6], Lennartz and Bovee [12] and Smith [21], but strengthens the hypothesis of Bakowska and JerkaDiadosz [2] according to which there must be some global control which monitors the overall size of the cell and the "bigger" parts of the oral ciliature as well as a local control which monitors the inner proportions of each membranelle in concert with the dimensions of the whole oral apparatus. Rickards and Lynn [18] reported that in B. [aponicum, differently from all other oral features, the larger is the body size, the smaller is the intermembranellar

348 . G. Rosati, A. Giari and N. Ricci

Ultrastructure of O. bifaria Giants . 349

distance. This does not seem to be the case in O. bifaria where, however, the intermembranellar spacing is the only oral feature which does not vary. So Fenchel's assumption [5,6] that the spacing of oral ciliature determines the minimum size particles retained but not the "selection" of different sized particles is further confirmed. In the case of Pi and Pe, it is somewhat different: while in the normal cells they consist of one and two rows of kinetosomes, respectively, in the giants they undergo a different process, the structure of the Pi being kept constant and that of the Pe being changed to 3 ciliary rows. Such a finding indicates that the two ciliary organelles, although lying elose to each other, are rather different as to their morpho-physiological significance, as already indicated in Paraurostyla [2 ]. The only oral structure showing a variation in the relative proportions of its dimensions is the cytopharynx which increases in length more than in width. This fact may be indicative of a certain degree of local regulation of a single structure according to its function. If the role of the cytopharynx is actually that of leading the food organisms deeply into the cytoplasm its increase in length is necessary in giant cells. Its widening, on the contrary, may be achieved just at the very moment of the ingestion of the prey with the help of the exceptionally conspicuous rnicrotubular bundles present in the cytoplasm surrounding the cytopharynx.

References 1 Ammerman D. (1985): Species charact erization and speciation in StylonychialOxytricha group (Ciliata, Hypotrichida, Oxytrichidae) . Atti Soc, Tose. Sei. Nat. Mem., Serie B, 92, 15-27. 2 Bakowska J. and ]erka-Dziadosz M. (1980): Ultrastructural aspect of size dependent regulation of surface pattern of cornplex ciliary organelle in a protozoan ciliate. J. Embryol, Exp. Morph., 59, 355-375. 3 Corliss J. O. (1979): The ciliated protozoa. Pergamon Press, Oxford. 4 Dogiel V. A. (1929): Polymerisation als ein Prinzip der progressiven Entwicklung der Protozoen. Biol, ZentralbL, 49, 451-469 . 5 Fenchel T. (1980): Suspension feeding in ciliated protozoa: structure and function of feeding organelles. Arch. Protistenk., 123, 239-260. 6 Fenchel T. (1980 ): Suspension feeding in ciliated protozoa functional response and particle size selection. Microb. Ecol.,

6, 1-11. 7 Giese A. C. (1938): Cannib alism and giantism in Blepharisma. Trans. Amer. Micros. Soc., 57, 245-255 . 8 Grimes G. W. and Adler ]. A. (1976): The structure and

development of the dor sal bristle complex of Oxytricha [allax and Stylonychia pustulata. J. Protozool. , 23, 135-143. 9 Kaul N., Sapra G. R. and Dass C. M. (1982): Intracellular digestive channel system in the ciliate Stylonychia mytilus Ehrenberg. Arch. Protistenk. , 126, 455-474. 10 ]erka Dziadosz M. (1976): The proportional regulation of cortical structures in a hypotrich ciliate Paraurostyla weissei. J. Exp. ZooL, 195, 1-14. 11 ]erka Dziadosz M. and Golinska K. (1977): Regulation of ciliary pattern in ciliates. ]. Protozool. , 24, 19-26. 12 Lennartz D. C. and Bovee E. C. (1980): Induetion of rnacrostome formation in Blepbarisma americanum (Suzuki, 1954) by alphatocopheryl succinate. Trans. Amer. Microsc. Soc., 99,310-31 7. 13 Polyanski G. 1. and Raikov 1. B. (1976): Polymerization oligomerization phenomenon in protozoan evolution. Trans. Amer. Microse. Soc., 95, 314-326. 14 Ricci N. (1981): Preconjugant cell interactions in Oxytricha bifaria (Ciliata, Hypotrichida): a rwo-step recognition process leading to cell fusion and the induction of meiosis. In: Horgen P. A. and O'Day D. H. (eds.): Sexual interaction in eukaryotic microbes. Academic Press, New York. 15 Ricci N., Banchetti R. e Cetera R. (1980): Messa a punto di una tecnica di cultura per il ciliato ipotrico Oxytricha bifaria Stokes. Atti Soc. Tose. Sc. Nat. Mem., 87, 211-218. 16 Ricci N. and Riggio D. C. (1984): Cannibals of Oxytricha bifaria (Ciliata, Hypotrichida). 1. A crowding-dependent cell differentiation. J. Exp. ZooL, 229, 339-347. 17 Ricci N., Verni F. and Rosati G. (1985): The cyst of Oxytricha bifaria (Ciliata Hypotrichida) . 1. Morphology and significance. Trans. Am. Micros. Soc., 104, 70-78. 18 Rickards J. C. and Lynn D. H. (1985): Can ciliates adjust their ultramembranellar spacing to prey size? Trans. Amer. Microse. Soc., 104, 333-340. 19 Riggio D. c., Ricci N., Banchetti R. and Seyfert H. M. (1987): Cannibals of Oxytricha bifaria (Ciliata, Hypotrichida). Macro- and micro-nuclear DNA content. Can. J. ZooL,65, 847-851. 20 Santangelo G. and Barone E. (1987): Results on cell volume, growth rate and DNA variation in a ciliated protozoon. J. Exp. Zool., 243, 401-407. 21 Smith H. E. (1982): Or al apparatus structure in the carnivorous macrostomal form of Tetrahymena vorax. J. Protozool., 29, 616-627. 22 Wicklow B. J. (1981): Evolution within the order Hypotrichida (Ciliophora, Protozoa): ultrastructure and morphogenesis of Thigmokeronopsis jahodai (n. gen., n. sp.); phylogeny in the Urostylina (jankowski, 1979). Protistologica, 17, 331-351. 23 Wicklow B. J. (1982): The Discocephalina (n. subord .): ultrastructure, morphogenesis and evolutionary implications of a group of endemie marine interstitial hypotriehs (Ciliophora, Protozoa). Protistologica, 18, 293-330. 24 Wirnsberger E., Foissner W. and Adam H. (1986): Biometrie and morphogenetic comparison of the sibling species Stylenychia mytilus and S. lemnae including a phylogenetic system for the oxytrichid s. Arch. Protistenk., 132, 167-185 .

Key words: Oxytricha bifaria - Cannibals - Giants - Ultrastrueture - Differentiation Giovanna Rosati, Dipartimento di scienze dell'ambiente e del territorio, via A. Volta 4, 56100 Pisa, Italia <4

Figs. 5-10. TEM micrographs concerning the buccal apparatu s. - Fig. 5. section through the proximal AZM of anormal O. bifaria.Fig. 6. the same region of a giant. - Fig. 7. the cytopharynx of anormal cell. - Fig. 8. cross section showing a portion of the parorales in anormal cell. - Fig. 9. the parorales in a giant. - Fig. 10. the cytopharynx of a giant. Pe = parorale externe; Pi = parorale interne. Scale bars represent 2 um,