The unicellular ciliate Chilodonella steini has a well-defined flat and ciliated ventral field. During divisional morphogenesis two sets of new contractile vacuole pores (CVPs) are formed on this field. During final pattern formation some of these CVP primordia and the old parental set of CVPs are completely resorbed. Primary pattern of distribution of the CVP primoidia and final pattern of distribution of the matured CVPs manifest an intraclonal polymorphism.

From analysis of this polymorphism some features of mechanism(s) of CVP pattern determination are deduced:

  1. There is a strict, short-distance negative control of appearance of CVP primordia at sites of oral morphogenesis and around the ventral field.

  2. Certain indeterminacy of large-scale patterning of CVP primordia is observed over the area competent to yield CVP formation. However, within this area three longitudinal sectors with a high probability of occurrence of CVP primordia are alternated with sectors nearly deprived of their occurrence.

  3. Positive control of probability of occurrence and of specificity of location is found for certain CVP primordia. An interaction of mechanism of positioning on cellular longitudes and latitudes is proposed to account for these facts.

  4. The resorption of supernumerary CVP primordia does not alter the character of the global map of distribution of CVP primordia achieved during primary pattern formation. The primordia located at some latitudes persist, whereas others are resorbed at random. It is suggested that all CVP primordia which do not mature at the time of stabilization of divisional morphogenesis are resorbed. Thus the global map of CVPs distribution would result from the sum of the individual determinations of the fates of each CVP primordium, superimposed on an initial spatially non-uniform distribution of CVP primordia.

Ciliate are single-celled organisms with an ordered pattern of distribution of their organelles over the cell cortex. There is evidence that the mechanism of large-scale positioning of organelles is encoded in the ciliate genome (Heckmann & Frankel, 1968 ; Jerka-Dziadosz & Frankel, 1979) to ensure this species-specific order. However, in some ciliates the polymorphism exists. This may result from modification of the global map by effects of pre-existing organization of the ciliate cortex (Beisson & Sonneborn, 1965; Grimes, 1976; Ng & Frankel, 1977; Ng, 1979), either from the gradual regulation of patterns in the progeny of an abnormally patterned initial cell (Nanney, 1968), or from a probabilistic mode of determination of the number and of the location of organelles. This latter possibility is explored in this report on polymorphism of the disposition of contractile vacuole pores (CVPs) on the flat ventral surface of a ciliate, Chilodonella steini.

Any geometric description of the position of an organelle on the cell cortex requires specification of a coordinate system. The coordinate system projected over the cell surface allows one to measure absolute or relative distances from chosen reference points. At least two parameters are indispensable for the exact placement of the measured point on a two-dimensional surface. In a Cartesien system these parameters correspond to latitude and longitude (Frankel, 1979). In the polarized cortical layer of ciliates the evenly spaced meridional rows of ciliary basal bodies form the natural meridians for measuring longitudes of the organelle, with one of them -the stomatogenic meridian -serving as a reference. Nanney (1966 a, b, 1967, 1968) discovered certain cytogeometric rules of positioning of the CVP on longitudes in Tetrahymena. He gave a formal explanation of positioning of the CVP in a given cell as a consequence of specification of an inductive angle between the stomatogenic meridian and the area in which the CVP is found. The variability of positioning of the CVP between meridians is described in terms of a field angle, which defines the sector of the cell surface which may be competent to yield a CVP.

It is already established (Kaczanowska, 1974) that CVPs in Chilodonella cucullulus strain X form on three longitudinal sectors; the CVPs vary in number from 5 to 11, and their location is variable. CVPs are positioned at intersections of the longitudinal sectors with specific radii measured from a reference point, a site of stomatogenesis. If cells are microsurgically miniaturized (Kaczanowska, 1975), the proximal radius describes the exact placement of the anterior obligatory CVPs, while posterior CVPs are drastically reduced in number.

It is believed that an analysis of the polymorphism of CVP primordia and of CVPs distributions over the ventral field of the related species Chilodonella steini makes possible to delineate some general characters of a large-scale mechanism(s) operating in CVP-pattern determination. We begin by describing the number and distribution of CVP primordia and of CVPs in different specimens of Ch. steini at the same morphogenetic stage of division, when old CVPs and newly induced CVP primordia still coexist. Next we may investigate whether there are any similarities of the test patterns. The following issues are considered :

  1. Area occupied with CVP structures in all tested specimens apparently belong to regions competent to yield CVPs. Remaining areas may be either less competent to yield CVP formation, or are inhibited in CVP formation. If an area occupied with CVP primordia (or CVPs) is very sharply marked off from areas deprived of CVP primordia (or CVPs), this suggests that there are zones ‘forbidden’ from yielding CVP structures, i.e. they are under some form of negative control. If however CVP-competent areas gradually fade or merge with empty areas, this suggests that only positive controls are operating. It means that the appearance of CVP structures is most likely along certain meridians, with a gradual decrease in probability at more distant locations (Fig. 1, models A and B, left boxes).

  2. Grimes & L’Hernault (1979) and Frankel (1979) have established a different character of positioning operating on the longitudes and latitudes of a ciliate cell. Then the question arises whether or not these two supposed mechanisms act independently in determination of the position of a given organelle. It might be ascertained whether, in Ch. steini, the probability of occurrence of CVP structures and the degree of longitudinal dispersion remain constant within a given sector at all of its latitudes. In the event of some cooperation between the two putative mechanisms involved in positioning of CVP primordia, some CVP primordia within the same sector would be spatially more precisely located than others (Fig. 1, model C left box).

  3. In Ch. steini two sets of new CVP primordia appear during divisional morphogenesis while the old set of parental CVPs still persists. The primary patterns of the distribution of CVP primordia is then changed by the resorption of certain supernumerary CVP primordia. During final pattern formation, all supernumerary CVP primordia and the old set of parental CVPs completely disappear. The comparison of the primary and final patterns is made to test whether resorption of some CVP primordia may modify the general character of their distribution. Does it expand the ‘forbidden’ areas by eliminating CVP primordia at the boundaries or does it occur throughout the CVP areas merely decreasing their number but not affecting the boundaries of the CVP zone? If it is not at random this proves that final pattern determination takes place in two steps: first a broad outline of CVP areas, second some modification of these areas by the process of CVP primordia resorption. The alternative possibility, of random resorption of some of CVP primordia over the ventral field, would, if it occurred, still leave open the question of the reason of this randomness. The two different models of final pattern regulation subsequent to each of the three types of initial pattern establishment are depicted schematically in Fig. 1 (right boxes).

Fig. 1.

Theoretical models of CVP primordia and CVPs distribution within a zone competent to yield CVP formation in Chilodonella steini. Boxes represent an entire area of the ventral field. Black circles represent CVP primordia (left boxes), or matured CVPs (right boxes). White circles mark resorbed CVP primordia absent in final patterns (right boxes). Model A. Negative control, or inhibition of CVP primordia appearance in the extreme ‘forbidden’ zones. Sharp boundaries (solid lines) between CVP competent zone and CVP deprived zones. Primordia are randomly distributed within CVP competent zone. This might lead, following resorption of some CVP primordia, to either of two alternatives: Al -an attenuation of the CVP competent zone (indicated by transposition of solid lines, while dashed lines mark positioning of original boundaries), or A2 -maintenance of the original boundaries with random selection of CVP primordia for resorption. Model B. Probabilistic model of CVP primordia distribution along a preferred meridian (heavy line), but with a high dispersion of placement of CVP primordia. Delineated by dashed lines external zones mark zones of a very low probability of CVP primordia occurrence. Following resorption there is either Bl -an attenuation of the original dispersion (internal dashed lines), or B2 -maintenance of the original dispersion. Model C. Positive control of placement of CVP primordia at the intersection of two coordinates (crossing heavy lines). This might be followed by either: Cl -maintenance of CVP primordia at that intersection (dashed circle), or C2 -by a random selection of CVPs.

Fig. 1.

Theoretical models of CVP primordia and CVPs distribution within a zone competent to yield CVP formation in Chilodonella steini. Boxes represent an entire area of the ventral field. Black circles represent CVP primordia (left boxes), or matured CVPs (right boxes). White circles mark resorbed CVP primordia absent in final patterns (right boxes). Model A. Negative control, or inhibition of CVP primordia appearance in the extreme ‘forbidden’ zones. Sharp boundaries (solid lines) between CVP competent zone and CVP deprived zones. Primordia are randomly distributed within CVP competent zone. This might lead, following resorption of some CVP primordia, to either of two alternatives: Al -an attenuation of the CVP competent zone (indicated by transposition of solid lines, while dashed lines mark positioning of original boundaries), or A2 -maintenance of the original boundaries with random selection of CVP primordia for resorption. Model B. Probabilistic model of CVP primordia distribution along a preferred meridian (heavy line), but with a high dispersion of placement of CVP primordia. Delineated by dashed lines external zones mark zones of a very low probability of CVP primordia occurrence. Following resorption there is either Bl -an attenuation of the original dispersion (internal dashed lines), or B2 -maintenance of the original dispersion. Model C. Positive control of placement of CVP primordia at the intersection of two coordinates (crossing heavy lines). This might be followed by either: Cl -maintenance of CVP primordia at that intersection (dashed circle), or C2 -by a random selection of CVPs.

Data reported here are taken as evidence that:

  1. There is a very strict negative control of appearance of CVP primordia in some ‘forbidden’ areas. These areas are confined to the sites of stomatogenesis and to the border of the ventral field.

  2. In the remaining areas competent to yield CVP primordia, three sectors of high probability of occurrence of CVP primordia manifest different widths.

  3. Some CVP primordia are spatially much more precisely determined than others.

  4. Resorption of the supernumerary CVP primordia during final pattern formation does not alter the character of CVP distribution over the ventral field. However, certain CVPs, which are invariant elements of pattern, are never resorbed.

A clone of Chilodonella steini (Ciliata, Kinetofragmophora; Radzikowski & Golembiewska, 1977) line 237/10 that did not self in the immaturity period (Kaczanowski, Radzikowski, Malejczyk & Polakowski, 1980) was isolated from one exconjugant. Six months later, one subclone was reinitiated. All preparations of this subclone were made during a one-month period. Other cells tested were derived from the same subclone about 10 months later, when at least some cells entered into permanent selfing, with retention of old macronucleus (Kaczanowski et al. 1980).

The general characteristic of this species and the methods of culturing of the cells followed these of Radzikowski & Golembiewska (1977). The mean generation time of the cells varied from 12-19 h when they were maintained in a normal daily photoperiod and fed every second day.

Cells from 2-day mass clonal cultures were used for silver impregnation (method of Frankel & Heckmann, 1968). Well-silvered specimens in early division were selected for mapping and counting of their CVPs and CVP primordia if they were properly dorsoventrally embedded in gelatin and if all ciliary meridians were clearly distinguished. In 41 dividers of the subclone 237/10 immatured, protocols and maps were made of cortical parameters of 1842 CVP primordia and of 587 parental CVPs. If any coordinate system was tested (Figs. 6, 7, 8 and Table 1), all of the data were grouped and then statistically analysed (Sokal & Rohlf, 1969). In Fig. 8 three peaks were revealed in all three histograms. The significance of these results was tested for a given coordinate system by computing for every individual (n = 41) the difference in number of CVP primordia between a sector selected a priori in this coordinate system as having a high number of CVP primordia and neighbouring right and left sectors of the same width selected as having a low number of CVP primordia. In Table 1 in all specified sectors mean numbers and sd values of CVP primordia and of CVPs were calculated. To estimate the rate of decrease of total number of CVPs as compared to the total number of CVP primordia in specified sectors, the ratio of the number of CVPs occurring in a given sector of CVP primordia in it was calculated for every respective parental to anterior daughter, and parental to posterior daughter patterns of the same specimen. Then pooled data of the mean of all these ratios for every sector were compared with a Cochran and Cox test. The additional control cells fixed 10 months later involving only morphostatic cells were tested for reproducibility and the maintenance of the polymorphism of cells.

Table 1
graphic
graphic

Some statistical calculations (r correlation coefficients) have been made in the Center of Statistical Calculations of Warsaw University by Msc I. Wozniak.

(1) Divisional morphogenesis of Chilodonella steini

Divisional morphogenesis of Ch. steini conforms to the general scheme described for this genus (Radzikowski & Golembiewska, 1977).

Ch. steini is a flat asymmetric ciliate, with the ventral surface covered with ciliary meridians and subapical oral apparatus encircled by an oral ciliature. CVPs are distributed only over the ventral surface, but they never appear near the oral apparatus or at the margin of the ventral surface. In silvered specimens CVPs appear as round black circles between ciliary meridians (Fig. 2, arrows).

Fig. 2.

A ventral field of morphostatic Ch. steini. The CVPs are dispersed among the ciliary meridians (arrows).

Fig. 2.

A ventral field of morphostatic Ch. steini. The CVPs are dispersed among the ciliary meridians (arrows).

The first signs of approaching division are an increase of body size, differentiation of oral ciliary segments for the prospective posterior daughter cell (opisthe), and differentiation of two sets of new CVP primordia for the daughter cells as little spots or perpendicular slits near the left side of certain ciliary meridians (Fig. 3, arrows). Oral ciliary segments and one somatic segment (the so-called A-4 segment, Radzikowski, 1966; Kaczanowska, 1971) for the presumptive opisthe differentiate in the subequatorial region of the ventral field. Cells in this stage were selected for further study.

Fig. 3.

A ventral field of early dividing Ch. steini. In addition to old parental CVPs, new CVP primordia (arrows) are seen at the left sides of some ciliary meridians. The R, M and L sectors are marked off by dashed lines.

Fig. 3.

A ventral field of early dividing Ch. steini. In addition to old parental CVPs, new CVP primordia (arrows) are seen at the left sides of some ciliary meridians. The R, M and L sectors are marked off by dashed lines.

In the following stage of morphogenesis (Fig. 4), all oral segments of the presumptive opisthe and the ciliary segment A-4 begin to migrate by rotating around the centre of the oral region. While the ciliary meridians are passively transmitted into successive anterior daughter cells (protêts), in opisthes the enumeration of meridians of the postoral left part of the ventral field becomes altered due to a gain of one meridian from the A-4 segment. A-4 inserted backwards between a stomatogenic meridian (no. 1) and the meridian to its right (no. 2). When enumeration of meridians follows the rules for Tetrahymena (Elliott & Kennedy, 1973), the former meridian 1 now becomes meridian n, the former n becomes n-\ etc. (Figs. 4, 5). This slippage compensates for the usual loss of the extreme left meridian, which is not represented in the equatorial zone and is passed entirely to the proter. As a result of these migrations, the circumoral segments turn about 120° while the preoral segment is reversed and positioned anteriorly to them (Fig. 5). Some new CVP primordia change into long perpendicular slits, while others remain with no transformation (Fig. 4).

Fig. 4.

A ventral field of dividing Ch. steini at a stage, somewhat more advanced than that in Fig. 3. Segment A-4 is labelled with an arrow.

Fig. 4.

A ventral field of dividing Ch. steini at a stage, somewhat more advanced than that in Fig. 3. Segment A-4 is labelled with an arrow.

Fig. 5.

A ventral field of an advanced divider of Ch. steini. Morphogenetic movement of the oral segments is seen (heavy arrow). The new CVP primordia are undergoing transformation into the matured CVPs for daughter cells (arrows), while others are gradually discarded.

Fig. 5.

A ventral field of an advanced divider of Ch. steini. Morphogenetic movement of the oral segments is seen (heavy arrow). The new CVP primordia are undergoing transformation into the matured CVPs for daughter cells (arrows), while others are gradually discarded.

Fig. 6.

Diagram of the mean number of CVP primordia. per given intermeridional space counted from the stomatogenic axis for prospective proters of three different corticotypes. Corticotype 28 (n = 8) -dashed line, corticotype 27 (n = 11) -heavy line, and corticotype 26 (n = 6) -dashed and dotted line.

Fig. 6.

Diagram of the mean number of CVP primordia. per given intermeridional space counted from the stomatogenic axis for prospective proters of three different corticotypes. Corticotype 28 (n = 8) -dashed line, corticotype 27 (n = 11) -heavy line, and corticotype 26 (n = 6) -dashed and dotted line.

Fig. 7.

Diagram of the mean number of CVP primordia per given intermeridional space counted from the stomatogenic axis for prospective opisthes of three different corticotypes. Illustrative conventions and number of studied patterns as in Fig. 6.

Fig. 7.

Diagram of the mean number of CVP primordia per given intermeridional space counted from the stomatogenic axis for prospective opisthes of three different corticotypes. Illustrative conventions and number of studied patterns as in Fig. 6.

Fig. 8.

Diagram of the mean number of CVP primordia per given intermeridional space occurring both in prospective proter and opisthe in 41 specimens using the following reference coordinates: (a) the right extreme meridian, solid line; (b) the left extreme meridian, dashed and dotted line; (c) the stomatogenic axis, dotted line. Note that three different conventions of counting of ciliary meridians were used for ordering data about the CVP primordia deployment: convention (a), the most right ciliary meridian is designated as a ciliary meridian 1 and followed by 2, 3, etc.; convention (c), the most left ciliary meridian is designated as a ciliary meridian number 1 and followed in reverse direction by 2, 3, etc.; convention (c), furthest right postoral meridian is designated as a ciliary meridian number 1, to the right this meridian is followed with ciliary meridians 2, 3, etc., to the left this meridian is followed with ciliary meridians n, n-1,n-2, etc. This convention corresponds to that in Figs. 6 and 7. Three conventions of enumerations indicated on three abscissae. Ordinate: mean number of CVP primordia per intermeridional space.

Fig. 8.

Diagram of the mean number of CVP primordia per given intermeridional space occurring both in prospective proter and opisthe in 41 specimens using the following reference coordinates: (a) the right extreme meridian, solid line; (b) the left extreme meridian, dashed and dotted line; (c) the stomatogenic axis, dotted line. Note that three different conventions of counting of ciliary meridians were used for ordering data about the CVP primordia deployment: convention (a), the most right ciliary meridian is designated as a ciliary meridian 1 and followed by 2, 3, etc.; convention (c), the most left ciliary meridian is designated as a ciliary meridian number 1 and followed in reverse direction by 2, 3, etc.; convention (c), furthest right postoral meridian is designated as a ciliary meridian number 1, to the right this meridian is followed with ciliary meridians 2, 3, etc., to the left this meridian is followed with ciliary meridians n, n-1,n-2, etc. This convention corresponds to that in Figs. 6 and 7. Three conventions of enumerations indicated on three abscissae. Ordinate: mean number of CVP primordia per intermeridional space.

At a later stage of morphogenesis the old, parental oral apparatus (pharyngeal basket) is resorbed (Fig. 5) and very quickly two new oral apparatuses for the nascent daughters are formed: in situ for the proter and in the centre of a region of rotating ciliary segments for the opisthe. The CVP primordia gradually are transformed into the final round orifices in the middle of the intermeridional space, while the parental set of CVPs and some supernumerary new CVP primordia are resorbed.

In early dividers (Fig. 3) new CVP primordia are readily distinguished from parental CVPs by their shape and fine positioning (slits or dots in the left side of a ciliary meridian versus round bigger circles in the middle of the intermeridional space). In this stage, the future fission line is marked by the rupture of stomatogenic and left meridians. The right margin of the fission line may also be discerned as a small indentation of the dorsal surface. The distribution of CVP primordia may, therefore, be analysed separately in prospective proters and opisthes. In this stage the A-4 segment has not yet been added, and the old pattern of CVPs is perfectly preserved.

(2) Variability of the cortical patterns and of the total number of CVPs and CVPprimordia

Dividing specimens of Ch. steini manifested an array of corticotypes (i.e. total number of ciliary meridians) from 24 to 30 with corticotypes 27 and 28 in the majority of cells. In cells with corticotypes 26, 27 and 28 of the group tested for CVPs and CVP primordia, the distribution of the total dimensions of the ventral field were nearly identical. If the first (furthest right) stomatogenic meridian is designated as no. 1, it divides the whole set of ciliary meridians into right (nos. 2, 3, 4 etc.) and left ciliary meridians (nos. n, n-1, n-2, etc.). Cells of the same corticotypes often have different patterns of the total number of right and left meridians.

The total number of parental CVPs varies from 8 to 25 with a mean number of 14·7 ± 3·9 (n = 41). The newly formed sets of the CVP primordia included respectively 13 to 35 (mean 22·1 ± 5·0) for proters and 10 to 38 (mean 22·8 ± 6·2; n = 41) for opisthes. There is a significant (P = 0·01) difference between the total number of parental CVPs and the number of CVP primordia in descendants. It is deduced that some of the CVP primordia are discarded during formation of final pattern (about 34·5 % of the total number of CVP primordia).

There is no statistically significant correlation between the total number of parental CVPs and the number of CVP primordia in proters (r = 0·14) or in opisthes (r = 0·18). There is a slight positive correlation of the total number of the CVP primordia in proter and opisthe (r = 0·55 with P = 0·05).

The total number of the CVP primordia observed in particular intermeridional space is variable (from 0 to 10). Among 41 tested dividers no two cells had identical patterns of distribution of CVPs or CVP primordia.

Hence in Ch. steini there is a polymorphism of corticotypes, of right/left pattern of ciliary meridians, and of a number and disposition of CVP primordia and CVPs.

(3) Localization of the CVP primordia in early dividers, primary patterns

The maps of CVP primordia show that in all specimens there are zones strictly devoid of CVP primordia. These ‘forbidden’ zones include the margin of the ventral field, the regions of the parental oral apparatus and of the preoral ciliary segment, the area of the future oral primordium for the opisthe, and the region of the forming fission line.

The remaining surface of the ventral field is more or less competent to yield CVP primordia. However, these competent zones include longitudinal sectors of relatively high frequency of occurrence of CVP primordia. The right (R) sector follows the curvature of the right margin of the ventral field, but at some distance from it. The median (M) sector appears in the right half of the ventral field parallel to the main longitudinal axis of cells. This sector is roughly composed of two separate groups of CVP primordia for every daughter cell: one just posterior to the oral areas, which corresponds to the sites of CVP-1 described in the related species, Ch. cucullulus (Kaczanowska, 1974), and the second at the rear end of the ventral surface of the prospective daughter cell, corresponding to the posterior CVP-4 site of Ch. cucullulus. Finally, the left (L) sector appears in the middle portion of the left part of the ventral field. All of these sectors are diagramatically marked in Fig. 3.

Comparison of means of occurrence of CVP primordia in particular intermeridional spaces in cells of the same corticotypes (Fig. 6 for proters and Fig. 7 for opisthes) revealed that histograms for the proters and for the opisthes are very similar. The R, M and L sectors of frequent occurrence of CVP primordia alternate with four sectors of infrequent occurrence. The sectors at the left and right edges of the histograms belong to the ‘forbidden’ zones, while the sectors located between the R, M and L sectors, are characterized by a relative absence of the CVP primordia. The different corticotypes have a very similar variability of the mean frequency of CVP primordia. The longitudes of the CVP primary pattern might be computed for the whole ventral field by combining data for the prospective proters and opisthes for all 41 specimens. To test whether the observed R, M and L sectors might be also specified with reference to the boundaries of the ventral field, the positioning of all CVP primordia was then assessed in all specimens in terms:

  1. of the number of meridians from the right margin of the ventral field,

  2. or in terms of the number of meridians from the left margin of the ventral field,

  3. or in terms of the number of meridians from the stomatogenic axis.

In three histograms (Fig. 8) the R, M and L peaks are visualized in all three coordinate systems. Statistical tests were carried out to assess the significance of the peaks appearing within the three coordinate systems with respect to the neighbouring valleys. Particular intermeridional spaces were selected on an a priori basis as having either a high or low probability of appearance of CVP primordia. It was found that statistically significant results were obtained for the R and L sectors only if these were constituted of three intermeridional spaces, not one or two.

The three intermeridional spaces of the R peak had a significantly greater average number of CVP primordia than did the neighbouring valleys when these spaces were counted from the right margin of the ventral field; similar results were obtained for the left L peak with reference to the left margin. When the positions of the right and left peaks were enumerated with respect to the stomatogenic axis (Fig. 8, dotted line), these peaks were significant when compared to the proximal valleys (i.e. those between the stomatogenic axis and the peak) but, surprisingly, not with respect to the distal zones situated near the margins.

The M peak differed from the two other peaks in that it was made up of only a-single intermeridional space, and that it was significantly specified only with respect to the stomatogenic axis. This can be clearly appreciated by noting in Fig. 8 how much sharper this peak is in the spatial system keyed to the stomatogenic axis (dotted line) than in that keyed to the right (solid line) or left (dashed line) margins.

The simplest interpretation consistent with this analysis is that the M sector consists of only a single intermeridional space while the R and L sectors consist of three or, in the case of the L sector, probably more such spaces. The stomatogenic axis probably serves as the primary reference point for the M sector and may participate in specifying the R and L sectors; however, the ‘forbidden’ sectors at the two margins are significantly specified only in relation to the nearby cell margins.

Although, as mentioned earlier, the distribution of CVP primordia is probabilistic, and no two cells have an identical distribution, certain specific CVP primordia can be followed more reliably than others. We will here consider the two CVP primordia that may appear in the M sector, one of them (CVP-1) located a short distance posterior to the oral region, the other (CVP-4) situated not far from the posterior end of the nascent cells (Figs. 3, 5), as well as CVP-5, the anterior preoral CVP primordium in the R sector (these CVP primordia are numbered as in Ch. cucullulus stock X; Kaczanowska, 1974, 1975). The CVP-1 primordium was found in all but one of the 82 proter and opisthe examined patterns, and was always placed in the same intermeridional space with reference to the stomatogenic axis. Thus it occurred that CVP-1 primordium was a stable element of primary pattern of Ch. steini.

The CVP-4 primordium, on the other hand, was absent in 17 of the 82 patterns, preferentially in opisthes (but see also Fig. 4), and its location with respect to the stomatogenic axis was not absolutely fixed; in fact, all of the variation in the M sector is accounted for by variation in the placement of the CVP-4 primordium.

The preoral CVP-5 primordium failed to appear in only 2 out of the 82 daughter patterns. Its position, though not completely invariant, is mainly restricted to the interior intermeridional space within three ones constituting the R peak.

It thus appears that the level of indeterminacy in both the occurrence and the positioning of specific CVP primordia differs, both for primordia located at different latitudinal levels in the same sector (CVP-1 and CVP-4) and in different sectors at a somewhat similar latitude (CVP-1 and CVP-5).

(4) Comparison of the primary and final patterns of CVP distribution

In Ch. steini, only about 65·5 % of the CVP primordia persist in the final patterns. The remainder are resorbed. The question arises whether the probability of resorption is uniform over the whole competent area of occurrance of CVPs, or whether it is specifically confined to certain sectors (Fig. 1, right boxes).

Some decrease in the mean number of CVPs is observed in all of the specified sectors, both in the peaks of preferential occurrence of CVP primordia and in the valleys of relative absence of these primordia (Table 1). Further, the ratios of CVPs to CVP primordia did not differ significantly among sectors, as evaluated by the Cochran and Cox test (P = 0·05). This result strongly suggests the uniform resorption of CVP primordia over the whole CVP competent zone (corresponding to models of the alternatives nos. 2 among right boxes in Fig. 1).

But on the other hand, when specific CVPs (matured CVP-1, CVP-4 and CVP-5) were considered, CVP-1 was found to be absent at its expected site only in 1·6% of the specimens, CVP-5 was absent in 8% of the specimens, while the posterior CVP-4 was absent in 52·8 % ofthe cells. Thus the CVP-1 primordium tends to persist at a non-random fashion (Fig. 1 Cl model), while the CVP-4 primordia are much more readily resorbed.

These different data are taken as evidence for a generally uniform resorption of the total number of CVP primordia that is superimposed upon, and independent of, the spatially non-uniform probability of formation of CVP primordia and of their persistence.

In Chilodonella steini, CVP primordia may occur near any ciliary meridians except those in certain ‘forbidden’ areas. The borders of the ventral field and the site of stomatogenesis were used here as a priori reference points for CVP positioning on longitudes. Absolute absence of CVP primordia in these areas is consistent with a hypothesis of a short-distance inhibitory effect of the site of stomatogenesis and of the boundaries of the ventral field on the competence to yield CVP primordium formation.

Every meridian on the remainder of the ventral field is able to support CVP formation, although the probability of this event is much higher in three longitudinal sectors. From this result three conclusions may be drawn: (a) CVP organellogenesis is not restricted to particular meridians but rather to certain territories, (b) there is some indeterminacy in the large-scale mechanism of specification of these territories, as CVP primordia sometimes form outside of the territories and since even within the territories the disposition of CVP primordia is variable, and (c) there is some periodicity of longitudes of high probability of the occurrence of CVP primordia in a form of the R, M and L sectors alternated with sectors of low probability of CVP primordia occurrence. The right and left sectors of a high probability of occurrence of CVP structures cover more than one intermeridional space. In terms of Nanney’s formalism (1966b) they represent a broad field angle. The median sector, however, is limited to one intermeridional space.

A virtual stability of occurrence and of localization of the CVP-1 primordium with respect to the stomatogenic meridians, which undergo cortical slippage in every generation of opisthes, indicates that the stomatogenic axis is not inherited by the structural identity of a particular meridian, but as a territory in which stomatogenesis takes place. The position of the M sector, and especially the CVP-1 primordium is determined in relation to this territory.

At least CVP-1 primordium placement is determined much more specifically than the placement of the other CVP primordia. This suggests that along a given sector of high probability of appearance of CVP primordia, there exists, at some latitudes a spatial constraint on the CVP placement along longitudes (Fig. 1, model C). Thus a cytogeometric model of CVP distribution in Chilodonella (Kaczanowska, 1974) may result from some cooperation of mechanisms of positioning on longitudes and on latitudes, perhaps in a form of a mosaic of nodes of increased specificity of CVP positioning.

Ultrastructural investigations (Kaczanowska & Moraczewski, in preparation) indicate that during late division stages some CVP primordia are very advanced in their differentiation, while other neighbouring ones are still in an early stage of development. This asynchronous development of individual CVP primordia and next resorption of part of them, while others persist evidence a very local character of completion of CVP organellogenesis, which is different from the global character of assessment of CVP-competent territories observed at the cellular level of organization. This statement is consistent with a distinction made between large-scale and short-range mechanisms of patterning in ciliates (Frankel, 1979).

The final pattern of CVPs results from the spatially uniform resorption of about 35 % of the total number of CVP primordia. Resorption of the supernumerary CVP primordia does not modify the global map of CVP distribution over the ventral field. However, CVP primordia occurring at particular sites (e.g. CVP-1) are positively selected to persist. This juxtaposition of positive selection of certain of the CVP primordia and a randomness of the global fates of CVPs observed at the cellular level of organization might be understood by assuming an early structural maturation of the CVP primordia positioned at sites of increased specificity of CVP positioning. They might be sufficiently developed at the critical period of divisional morphogenesis (Kaczanowska & Kiersnowska, 1976) of Chilodonella to escape resorption.

Thus the global map of CVPs distribution in Ch. steini would result from the sum of the individual determinations of the fates of each CVP primordium, superimposed on an initially spatially non-uniform distribution of CVP primordia.

In terms of the set of theoretical models of CVPs distribution on the ventral surface of Ch. steini (Fig. 1) presented here data are consistent with model A applied to dissect a CVP competent zone out of ‘forbidden’ zones at the sites of stomatogenesis and around the border of the ventral field. On remaining zone there are three preferred sectors of CVP primordia occurrence with certain dispersion of their placement (Model B). However, along these sectors the positive control of placement of certain CVP primordia is also established (perhaps consistent with Model C). Resorption, though globally random, involves a positive selection of at least CVP-1 primordium (Model Cl).

I am most grateful to Dr Joseph Frankel for extensive discussions, helpful suggestions and criticisms in the development and final shaping of this manuscript. The author would like to thank Dr Maria Jerka-Dziadosz, Dr Krystyna Goli ń ska and Dr Andrzej Kaczanowski for critical reading of the draft of this manuscript.

This work is partially supported by a research grant of the Polish Academy of Sciences P.A.N.-II. 1.3.7.

Beisson
,
J.
&
Sonneborn
,
T. M.
(
1965
).
Cytoplasmic inheritance of the organization of the cell cortex in Paramecium aurelia
.
Proc. natn. Acad. Sci., U.S.A
.
53
,
275
282
.
Elliot
,
A. M.
&
Kennedy
,
J. R.
(
1973
).
Morphology of Tetrahymena
.
In Biology of Tetrahymena
(ed.
A. M.
Elliot
), pp.
57
87
.
Stroudsbury, Pa
:
Dowden, Hutchinson and Ross Inc
.
Frankel
,
J.
(
1979
).
An analysis of cell-surface patterning in Tetrahymena
.
In Determinants of Spatial Organization
(ed.
S.
Subtelny
&
I. R.
Konigsberg
), pp.
215
246
.
New York
:
Academic Press
.
Frankel
,
J.
&
Heckmann
,
K.
(
1968
).
A simplified Chatton-Lwoff silver impregnation procedure for use in experimental studies with ciliates
.
Trans. Amer, microsc. Soc
.
87
,
317
321
.
Grimes
,
G. W.
(
1976
).
Laser microbeam induction of incomplete doublets of Oxytricha fallax
.
Genet. Res. Cambridge
27
,
213
226
.
Grimes
,
G. W.
&
L’Hernault
,
S. W.
(
1979
).
Cytogeometrical determination of ciliary pattern formation in the hypotrich ciliate Stylonychia mytilus
.
Devl Biol
.
70
,
372
295
.
Heckmann
,
K.
&
Frankel
,
J.
(
1968
).
Genic control of cortical pattern in Euplotes
.
J. exp. Zool
.
168
,
11
38
.
Jerka-Dziadosz
,
M.
&
Frankel
,
J.
(
1979
).
A mutant of Tetrahymena thermophila with a partial mirror image duplication of cell surface pattern. I. Analysis of phenotype
.
J. Embryol. exp. Morph
.
49
,
167
202
.
Kaczanowska
,
J.
(
1971
).
Studies on topography of the cortical organelles of Chilodonella cucullulus. (O.F.M.). III. Morphogenetic movements, regional multiplication of kinetosomes and cytokinesis in normal dividers and after phenethyl alcohol treatment
.
Acta Protozool
.
8
,
84
103
.
Kaczanowska
,
J.
(
1974
).
The pattern of morphogenetic control in Chilodonella cucullulus
.
J. exp. Zool
.
187
,
47
62
.
Kaczanowska
,
J.
(
1975
).
Shape and pattern regulation in regenerants of Chilodonella cucullulus (O.F.M
.).
Acta Protozool
.
13
,
343
360
.
Kaczanowska
,
J.
&
Kiersnowska
,
M.
(
1976
).
Thermosensitivity of pattern in a ciliate Chilodonella cucullulus
.
J. exp. Zool
.
196
,
135
148
.
Kaczanowski
,
A.
,
Radzikowski
,
S.
,
Malejczyk
,
J.
&
Polakowski
,
I.
(
1980
).
Study on intraclonal pairing in Chilodonella steini. Evidence of abortive conjugation
.
J. exp. Zool
.
213
,
262
269
.
Nanney
,
D. L.
(
1966a
).
Cortical integration in Tetrahymena. An exercise in cytogeometry
.
J. exp. Zool
.
161
,
307
318
.
Nanney
,
D. L.
(
1966b
).
Corcicotype transmission in Tetrahymena
.
Genetics
54
,
955
968
.
Nanney
,
D. L.
(
1967
).
Cortical slippage in Tetrahymena
.
J. exp. Zool
.
166
,
163
169
.
Nanney
,
D. L.
(
1968
).
Cytogeometric integration in the ciliate cortex
.
Ann. N.Y. Acad. Sci
.
193
,
14
28
.
Ng
,
S. F.
(
1979
).
The precise site of origin of the contractile vacuole pore in Tetrahymena and its morphogenetic implications
.
Acta Protozool
.
18
,
305
312
.
Ng
,
S. F.
&
Frankel
,
J.
(
1977
).
180°-rotation of ciliary rows and its morphogenetical implications in Tetrahymena pyriformis
.
Proc. natn. Acad. Sci., U.S.A
.
74
,
1115
1119
.
Radzikowski
,
S.
(
1966
).
Study on morphology, division and postconjugation morphogenesis in Chilodonella cucullulus (O.F. Mueller)
.
Acta Protozool
.
4
,
90
96
.
Radzikowski
,
S.
&
Golembiewska
,
M.
(
1977
).
Chilodonella steini, morphology and culture method
.
Protistologica
13
,
381
389
.
Sokal
,
R. R.
&
Rohlf
,
F. J.
(
1969
).
Biometry
.
San Francisco
:
Freeman, Cooper and Co
.