Abstract
Paramecium tetraurelia normally resorbs the preexisting oral apparatus (and develops a new one) during sexual reproduction. Violation of this rule was found in amicronucleate cell lines. These cell lines generated chains of two cells (homopolar tandems) at a low frequency, as a result of incomplete binary fission during a transient growth depression period following emicronucleation. In autogamous chains, the proter resorbed the pre-existing oral structures, while some of the opisthes retained them. The oral structures in the opisthes of the chains were unusually close to the opisthes’ anterior end. The ectopic location of these oral structures might account for their retention, formally understood in terms of the theory of positional information. It is suggested that nongenic factors, likely involving components of the rigid cortical matrix, are involved in the fixation of positional values.
Introduction
The oral apparatus of ciliated protozoa has been extensively employed in studies of control of development in unicells (e.g. Frankel, 1984; Frankel et al. 1984; Golinska, 1978, 1984; Grimes, 1982; Hanson, 1955, 1962; Jerka-Dziadosz, 1974, 1983; Sonneborn, 1963,1970a; Tam & Ng, 1987a; Tartar, 1961; Williams & Honts, 1987). While the development of a new oral apparatus during asexual and sexual reproduction is elucidated in detail in representative groups of ciliates, the resorption of the oral apparatus in some stages of the life cycle has been given little attention in general. The outstanding exception is the demonstration by microsurgery in some ciliates, notably Stentor, that a developing oral primordium regresses under the influence of a pre-existing oral apparatus (Tartar, 1961).
In Paramecium during sexual reproduction (autogamy or conjugation), the oral apparatus is resorbed and, simultaneously, a new one is produced (Fig. 1). Resorption of the oral apparatus is of developmental interest because this takes place invariably and exclusively during sexual reproduction. The consequence, viz. replacement of the pre-existing oral apparatus by a new one at the beginning of the clonal cycle, is of morphogenetic importance to the clone (see discussion in Ng & Tam, 1987).
Silver impregnation of a postautogamous micronucleate cell showing the normal oral apparatus with three oral membranelies (quadrulus, dorsal and ventral peniculi), each comprising four basal body rows. The penicular basal body rows are compact, whereas those of the quadrulus spread out in the anterior part. The membranelles are enclosed in a buccal cavity deep in the cytoplasm.
Silver impregnation of a postautogamous micronucleate cell showing the normal oral apparatus with three oral membranelies (quadrulus, dorsal and ventral peniculi), each comprising four basal body rows. The penicular basal body rows are compact, whereas those of the quadrulus spread out in the anterior part. The membranelles are enclosed in a buccal cavity deep in the cytoplasm.
Amicronucleate paramecia are of interest in that, during sexual reproduction, they fail to develop a new oral apparatus, but resorb the pre-existing one (Ng & Mikami, 1981; Ng & Newman, 1984). However, in some amicronucleate cell lines, oral structures persisted in about 1 % of the cells going through autogamy (Ng, in preparation). In addition, the present study documents another type of amicronucleates exhibiting an even higher frequency of retention of oral structures after autogamy. These are chains of two cells arising from incomplete binary fission in amicronucleate cultures. The unusual configuration of these chains, the abnormal location of the oral structures in them and the disparity in the fate of the oral structures in the anterior and posterior members of the chain together allow us to gain understanding in the control of resorption of the oral apparatus during sexual reproduction.
Materials and methods
Cells and culture
Four stocks of Paramecium tetraurelia were used in the present study: stock 51, the standard stock; stock d4-94, isogenic with stock 51 except for the pwA1 gene (Kung, 1971); stock ‘bp’, an F2 derivative (bu1/bu1 ; pwA1/pwA1) of a cross between d4-94 and the mutant ‘buccalless 1’ (Tam & Ng, 1987b); stock d4-78, isogenic with stock 51 except for the chi gene, producing cells with varying numbers of micronuclei (Sonneborn, 1975).
Cells were cultured in phosphate-buffered (pH ≃7) cerophyl medium (2·5 gm l-1) bacterized with Enterobacter aerogenes, supplemented with stigmasterol (5 mg l-1). For handling of paramecia, the methods of Sonneborn (1950, 1970b) were followed.
Generation of amicronucleate cell lines and collection of chains
Amicronucleate cell lines were initiated by removal of the two micronuclei, one after the other with an intervention of several fissions, from cells of young clonal ages (around 20 fissions), with a micromanipulated injection set-up as described by Ng (1981). Four such amicronucleate cell lines of independent clonal origins were obtained from stock 51, three from d4-94 and one from stock bp. In addition, two amicronucleate cell lines were obtained from d4-78 by selecting cells of smaller sizes from a postautogamous culture, as described in Ng & Newman (1982). After their initiation, the amicronucleate cell lines were cultured in 27°C in glass depression slides. Duplicate sets of cultures were kept in 15°C. Amicronuclearity was ascertained by staining samples of cells with aceto-orcein (Beale & Jurand, 1963), without prior osmium fixation.
During the initial growth depression period which usually lasted <40 fissions (Ng & Mikami, 1981; Ng & Tam, 1987), the amicronucleate cell lines produced chains of two cells arising from incomplete binary fission. These chains arose usually during the early phase of growth depression, and in low frequency (<1 %). These chains were collected from log and late-log cultures in 27°C, and sometimes also from those in 15°C, and silver impregnated for cytology. Most of these chains collected had undergone autogamy, attested by complete fragmentation of the macronucleus.
Cytology
Cells were silver impregnated (Chatton & Lwoff, 1936; Corliss, 1953; with modifications) and studied under ×1000 ph ase-contrast optics. Cell length (a) and the distance of the anterior end of the oral membranelles from the anterior end of the cell (b), were measured with an ocular micrometer. The ratio b/a furnished an assessment of the latitudinal location of the oral apparatus, in relation to the length of the cell.
Statistics
Comparisons of two percentages were done by 2×2 G-test, with William’s correction when n<30 (Sokal & Rohlf, 1981). Comparisons of the means of the latitudinal locations of oral structures (i.e. the b/a ratio, see above) were done by Student’s t-test.
Results and discussion
In Paramecium, the new oral apparatus arising in an equatorial region (in the posterior-right wall of the vestibule) descends into the opisthe as constriction of the fission furrow bissects the cell into two (Jones, 1976; Shi, 1980). In amicronucleates, chains of two cells, frequently bent into a V-shape, arose as a result of incomplete constriction of the fission furrow during binary fission. This was related to, if not caused by, defective stomatogenesis in amicronucleates in growth depression.
In most of these chains, the opisthes’ oral structures were found close to the fission zone, but anterior to the fission furrow. A buccal cavity was missing in these oral structures. The oral membranelles were frequently broken up; in these, the cytopharyngeal membrane and the ribbed wall, when present, were all exposed on the cell surface (Fig. 2). In others, the new oral structures of the opisthe lay posterior to the fission furrow; sometimes the buccal cavity was present and the opisthe’s oral apparatus was functional in feeding (Figs 3,4). The frequency of the presence of oral structures in the opisthes was given in Table 1 (‘Vegetative’). In a few chains, the opisthe’s oral structures were split into two parts which lay anterior and posterior to the fission furrow (Figs 5,6).
Silver impregnation of chains in amicronucleate cultures (none from stock d4-78) showing the deployment of oral structures in vegetative cells (Figs 2–6) and in postautogamous cells (Figs 7–11). The broken line marks the fission furrow; oral structures of opisthes are marked with arrows. For legends see page 592.
Fig. 2. Oral structures of the opisthe found anterior to the fission furrow. The opisthe’s oral metnbranelles are broken in the middle into two portions, probably as a result of the bending of the opisthe. The portion on the (cell’s) right is rotated 180° and becomes a mirror image of the portion on the left.
Figs 3, 4. Oral structures of opisthes posterior to the fission furrow, exposed on the surface in Fig. 3, but complete with the buccal cavity in Fig. 4.
Figs 5, 6. Two chains showing the opisthe’s oral structures split into two parts lying anterior and posterior to the fission furrow. A and B are two focal levels of the same cell. In Fig. 5 the proter is viewed dorsally and its oral structures are seen by focusing through the cell.
Fig. 7. An exceptional postautogamous chain showing persistence of both oral structures of the proter (A) and those of the opisthe anterior to the fission furrow (B, arrow). A and B are two focal levels through the cytoplasm. The proter is viewed dorsally and the oral structures are seen by focusing through the cell. The direction of bending of the middle part of the proter’s quadrulus is also abnormal (to the cells right instead of to the left, c.f. Fig. 1).
Figs 8–11, Postautogamous chains with oral structures posterior to the fission furrow persisting in the opisthes. These oral structures are mostly closer to the fission furrow than usual (Figs 8, 10), and rarely close to the posterior end of the opisthe (Fig. 9). The oral structure in Fig. 10 is complete with a buccal cavity leading posteriorly to the cytopharynx and food-vacuole-forming region (arrow). Fig. 11 shows an opisthe that has become detached from the chain, viewed from the left, in two focal levels (A,B). The oral apparatus is anteriorly displaced and the outline of the buccal cavity can be seen (Fig. 11B, arrow). Food vacuoles are present.
Silver impregnation of chains in amicronucleate cultures (none from stock d4-78) showing the deployment of oral structures in vegetative cells (Figs 2–6) and in postautogamous cells (Figs 7–11). The broken line marks the fission furrow; oral structures of opisthes are marked with arrows. For legends see page 592.
Fig. 2. Oral structures of the opisthe found anterior to the fission furrow. The opisthe’s oral metnbranelles are broken in the middle into two portions, probably as a result of the bending of the opisthe. The portion on the (cell’s) right is rotated 180° and becomes a mirror image of the portion on the left.
Figs 3, 4. Oral structures of opisthes posterior to the fission furrow, exposed on the surface in Fig. 3, but complete with the buccal cavity in Fig. 4.
Figs 5, 6. Two chains showing the opisthe’s oral structures split into two parts lying anterior and posterior to the fission furrow. A and B are two focal levels of the same cell. In Fig. 5 the proter is viewed dorsally and its oral structures are seen by focusing through the cell.
Fig. 7. An exceptional postautogamous chain showing persistence of both oral structures of the proter (A) and those of the opisthe anterior to the fission furrow (B, arrow). A and B are two focal levels through the cytoplasm. The proter is viewed dorsally and the oral structures are seen by focusing through the cell. The direction of bending of the middle part of the proter’s quadrulus is also abnormal (to the cells right instead of to the left, c.f. Fig. 1).
Figs 8–11, Postautogamous chains with oral structures posterior to the fission furrow persisting in the opisthes. These oral structures are mostly closer to the fission furrow than usual (Figs 8, 10), and rarely close to the posterior end of the opisthe (Fig. 9). The oral structure in Fig. 10 is complete with a buccal cavity leading posteriorly to the cytopharynx and food-vacuole-forming region (arrow). Fig. 11 shows an opisthe that has become detached from the chain, viewed from the left, in two focal levels (A,B). The oral apparatus is anteriorly displaced and the outline of the buccal cavity can be seen (Fig. 11B, arrow). Food vacuoles are present.
As is well known, Paramecium resorbs the preexisting oral apparatus and develops a new one during sexual reproduction (autogamy or conjugation). Amicronucleate paramecia failed to develop a new oral apparatus but, with rare exceptions, resorbed the pre-existing oral apparatus (Ng & Mikami, 1981; Ng & Newman, 1984; Ng, in preparation). The situation with the amicronucleate chains in autogamy was different. The oral apparatus of the proter was almost invariably resorbed (except in 2 out of 437 cases); the same was the case with the (opisthe’s) oral structures found anterior to the fission furrow (Fig. 7). This low incidence of persistence of oral structures after autogamy is in agreement with the observation in postautogamous amicronucleate singlets (Ng, in preparation). Remarkably, however, oral structures posterior to the fission furrow, sometimes complete with a buccal cavity, persisted in many (Figs 8–11). Thus within a common cytoplasmic entity, the fate of the oral structures during autogamy differed according to whether they were anterior or posterior to the fission furrow. The incidence of this observation for eight amicronucleate cell lines, and also for amicronucleate chains arising in cultures of micronucleate stock d4-78 in autogamy, is recorded in Table 1 (‘Postautogamous’).
Do amicronucleate chains invariably retain the oral structures that are posterior to the fission furrow during autogamy? The answer is NO. This is clear from a comparison of the frequencies of the chains possessing oral structures posterior to the fission furrow in the vegetative state and after autogamy (Table 1). The null hypothesis that the two frequencies were identical was rejected in three of the four cell lines. Thus, only about 10–50 % of these oral structures were retained in the various amicronucleate cell lines. This shows that being posterior to the fission furrow does not provide a sufficient condition for retention of the oral structures.
The oral apparatus of the opisthe is newly developed during binary fission, as opposed to the one that persists with only partial redifferentiation in the proter (Shi, 1980). Can the disparity in the fate of the oral structures during autogamy in the proter and opisthe of the amicronucleate chains be understood in terms of their ontogeny? It appears not. This is simply because in normal singlets, the oral apparatus of the opisthe, one newly developed during binary fission, is invariably resorbed in autogamy. Furthermore, in a considerable proportion of the amicronucleate chains, the oral structures newly developed in binary fission failed to descend into the opisthe and came to be located anterior to the fission furrow in the posterior part of the proter. Nevertheless, apart from the one case (Fig. 7B), none of the proters of the chains retained these newly developed oral structures. Therefore, the retention of the opisthe’s oral structures in the amicronucleate chains in autogamy cannot be understood in terms of their nascent nature.
One interesting difference between the oral structures found in proters and opisthes is that the pattern of the proters’ oral membranelles in most cases was more normal compared to those in opisthes. This generates interest as to whether this might account for the resorption of the proters’ oral structures versus retention of those of the opisthes during autogamy. The resorption of old ciliature has been shown to be causally related to the presence of developing new ciliature in a hypotrichous ciliate, Urostyla; removal of the primordium of the developing ciliature prevented the resorption of the old ciliature (Jerka-Dziadosz, 1968). Moreover, in Paramecium, the development of a new oral apparatus during binary fission depends on the normality of the pre-existing oral apparatus (Hanson, 1955, 1962; Hanson & Ungerleider, 1973; Ng, unpublished data). In view of these observations, it is tempting to suggest that during autogamy of the amicronucleate chains, the relatively normal oral structures in the proter may permit the initiation of development of a new oral apparatus (which is only aborted later at a characteristic stage, Ng & Mikami, 1981; Ng & Newman, 1984). Such an initial development of the new oral apparatus in the proter may be sufficient to promote the resorption of the old oral apparatus. On the other hand, the more defective oral apparatus in the opisthe may not be able to initiate the development of the new oral apparatus and hence their old oral structures are frequently retained. Two observations, however, argue against this hypothesis. First, the oral apparatus of the proter is not entirely normal. The most conspicuous defect in many is the lack of the buccal cavity, so that these chains are nonfeeders. It is very likely that, in the first place, the cell giving rise to the chain lacks a buccal cavity, but attempts to divide using up reserves and thus producing a chain. This is supported by the observation that the chains harvested from the cultures are frequently autogamous, a sign of starvation, though other cells in the culture continue to divide. Hence, it is unlikely that such chains, not even in their proters, can initiate another round of oral development during autogamy. Second, the development of a new oral apparatus during autogamy differs from that during binary fission, in that it is less dependent on the normality of the preexisting oral apparatus (Hanson & Ungerleider, 1973), but more on the activity of the micronucleus (Ng & Tam, 1987). Working with Paramecium doublets, Hanson has shown that irradiation of one of the two oral apparatuses sometimes resulted in its elimination in the asexual progeny; however, when these progeny cells were induced to undergo autogamy within four fissions after the loss of the oral apparatus then a new oral apparatus could be reformed in the irradiated axis. This demonstrates that during autogamy the development of the new oral apparatus can be initiated in the absence of any visible old oral structures. We have further shown that cells possessing defective oral apparatuses, when induced to undergo autogamy, could generate more normal oral apparatuses, and we have concluded that the development of the oral apparatus in the sexual cycle is largely unrestrained by the pre-existing oral apparatus, but comes under the control of the micronucleus.
A striking feature of the oral apparatuses of amicronucleate chains concerns their ‘latitudinal locations’ (see Materials and methods). In stock 51 micronucleate singlets, the oral apparatus is normally located about 0·42 from the anterior end of the cell (Table 2). However, in the chains, the latitudinal locations of the opisthes’ oral structures differed significantly from this and were much closer to the anterior end of the opisthe. The converse was true in the proter of the chains; the oral structures were more remote from the anterior end of the cell than usual (except in proters of 51 amicronucleate no. 2). The closeness of the anterior and posterior oral structures to the fission furrow reveals a restriction in the growth of the cell on the two sides of the fission furrow during binary fission and, consequently, a change in the proportion of the cell body. Can the ectopic locations of the oral structures in the chains account for the disparity in their fate during autogamy? The simplest hypothesis is that the cell defines a developmental field between the two ends and assigns positional values in between (Wolpert, 1969; Frankel, 1974, 1984). When the latitudinal location of the oral structures is greater than a certain value, nominally = 0·4, they are resorbed during autogamy. This explains the resorption of oral structures in the normal location, as well as those that are found more posteriorly. The latter class includes the newly formed oral structures which, instead of being placed in the opisthe, become lodged with the posterior part of the proter anterior to the fission furrow in some of the amicronucleate chains; these oral structures, as we have noted, were almost invariably resorbed during autogamy. Conversely, oral structures in latitudinal locations less than 0·4, as found in the opisthes of the chains, stand a good chance of being retained during autogamy. This hypothesis can accommodate the observation that, in some cases, the oral structures in the opisthes of the chains are resorbed during autogamy, if the probability of resorption is a function of the magnitude of the oral latitudinal locations. This quantitative notion of the hypothesis can be tested by comparing the latitudinal locations of the oral structures that are retained in the opisthes of the chains after autogamy with those present in the vegetative chains. One expects that if this quantitative notion holds true then those oral structures that are preferentially retained would exhibit smaller latitudinal values. A preliminary analysis in this direction suggests that this could well be the case, but the small sample sizes involved preclude a firm statistical judgement to be made. This quantitative aspect is a fine point of the hypothesis, but is not vital to it, because retention of oral structures during autogamy may be possible once the location of the oral structures falls below a certain latitudinal value (nominally 0·4), and the probability of retention may not be strictly proportional to the magnitude of such values. A resolution of this fine point may be possible when more materials become available in the future. The understanding, however, is unlikely to come from studying amicronucleate cell lines, not only because of the rarity of the chains appearing within a particular amicronucleate cell line, but also because, as noted above, of the difficulty in controlling autogamy of the chains once they have arisen in the culture. The latter situation creates difficulty in harvesting a large number of vegetative chains for analysis.
The formal application of the theory of positional information to the understanding of retention of oral structures by the opisthes of the autogamous chains has interesting implications. The theory has been used to explain a number of morphogenetic phenomena in ciliated protozoa (reviewed by Frankel, 1974, 1984). Of interest is the demonstration of the regulative nature of developmental fields, as in the reversal of anteroposterior axis when two fields of opposite polarities are juxtaposed side by side, in Stentor (Tartar, 1961) and in several hypotrichs (see Frankel, 1984). The present observations on the chains show that juxtaposition of two developmental fields end to end entails different consequences. The two fields apparently maintain their own entities, each with its own set of positional values. The proter and opisthe, though cytoplasmically connected, are developmentally compartmentalized on the two sides of the fission furrow. This indicates that the maintenance of individuality of the two compartments must involve nongenic factors likely to be associated with the relatively rigid cortical matrix, including cell membranes and the fibrogranular layer underneath (epiplasm) (Hufnagel, 1986; Williams, 1986; see also Bardele, 1983). In terms of the theory of positional information, these structures would be playing a role in the fixation of positional values in the two compartments.
