Correlation between ventral surface structures and local degeneration of cilia during conjugation in Paramecium

Ciliated protozoa are advantageous as a model system for studying the mechanism of differentiation of the cell surface, because local structural and functional differentiation of the cortex is remarkable in these organisms (Sonneborn, 1977; Beisson, 1977; Frankel, 1979; Aufderheide, Frankel & Williams, 1980). Paramecium is one of the most extensively studied ciliates, and much is known about its cortical ultrastructure (Ehret & de Haller, 1963; Ehret & MacArdle, 1974; Jurand & Selman, 1969; Allen, 1971; 1978a) and morphogenetic processes (Sonneborn, 1963, 1970, 1975; Dippell, 1968; Chen-Shan, 1969,1970; Kaneda & Hanson, 1974). In these works, the highly ordered arrangement of cortical organelles and the processes of their multiplication and positioning during cell proliferation have been demonstrated. The cortex of Paramecium is formed of many cortical units which are composed of one or two cilia and their basal bodies and accessories, such as kinetodesmal fibres, some microtubule bands, and parasomal sacs. The specific arrangement of cortical units and other surface organelles forms cortical patterns which are maintained through cell division by cortical inheritance (Sonneborn, 1963; Beisson, 1977).

In addition to accumulation of information about the structural features of the cortex, local functional or morphogenetical differentiations have also been reported in Paramecium. Differential distribution of Ca²⁺ and K⁺ mechanoreceptor channels (Ogura & Machemer, 1980) and localization of a series of conjugation events (Hiwatashi, 1955, 1961; Vivier & Andre, 1961; Watanabe, 1978) are good examples of such surface specializations. No one has shown, however, that such functional differentiations are correlated with structural differentiation of Paramecium cortex, except for the feeding and excretion system and osmoregulatory system. It is well known that, for feeding and excretion and for osmoregulation, specific and conspicuous organelles, an oral apparatus and a cytoproct and two contractile vacuoles, are differentiated on a cell surface. Watanabe (1981) studied whether the local degeneration of cilia during sexual reproduction in Paramecium is correlated with a mosaic distribution of the two kinds of cortical units, units containing one cilium or two, but no direct correlation was observed.

The present work was performed to know if a direct correlation exists between the sites of ciliary degeneration during conjugation and the position of ventral surface structures, such as the suture line and the oral apparatus. For this purpose, aberrantly shaped cells were induced by treating the cells with adenine (Mishima, 1975). In these cells, the proportion of preoral and postoral regions in the cell is abnormal because of the aberrant location of the oral apparatus, but the mating reactivity is still preserved (Mishima, 1978).

Stock 27aG3 of Paramecium caudatum, mating type V in syngen 3, was used in the present work. Cells were grown at 25 °C in lettuce juice medium infected with Klebsiella pneumoniae one day before use (Hiwatashi, 1968). Aberrantly shaped cells were induced according to the method of Mishima (1975). Cells grown for 1 day in ordinary culture medium were harvested and resuspended at cell density of about 1500 cells/ml in newly prepared culture medium containing 2 mM adenine-HCl, and incubated at 25 °C for 24 h. The concentration of adenine (2 mM final concentration) was found to be the optimum for inducing abnormally shaped cells in P. caudatum. Although the optimum concentration is the same as for P. multimicronucleatum (Mishima, 1975), the maximum proportion of anomalies (about 10%) was much less than that of P. multi-micronucleatum (about 50%). Aberrantly shaped cells can be easily collected by micropipette and used for experiments.

For light microscopic observation, cells were fixed and stained with the Chatton-Lwoff silver impregnation technique (Corliss, 1953; Ng, 1976). Silver-impregnated animals were exposed to u.v. light soon after dehydration and permanent mounting. To see the localization of the ciliary degeneration, cells were fixed at 60-90 min after onset of mating reaction. At this time, some conjugating pairs can be seen. Methods for scanning electron microscopy were described previously (Watanabe, 1978,1981).

In the text, the terms right and left refer to the cell’s right and left sides relative to the suture.

In the normal process of cell proliferation, the new oral apparatus is formed from the right posterior portion of the old one, and then migrates through the surrounding cortex (see for review Kaneda & Hanson, 1974). During incubation of cells in the culture medium containing 2 mM adenine for 24 h, cell division processes are affected to different extents. Generally, migration of the oral apparatus towards its usual position in the prospective daughter cell, i.e. slightly posterior to the equator of the cell (Fig. 1), is inhibited. If the cell completes cell division, aberrantly shaped ‘proter’ (anterior fissont) and ‘opisthe’ (posterior fissont) cells are obtained. In the proter, the oral apparatus is located at or near the posterior end of the cells (Fig. 2). Therefore, the proter possesses a much wider anteroventral cortical field and a much longer anterior suture than those of a normal cell. The posterior portion of the proter cell is greatly reduced in size. In contrast, the oral apparatus of the opisthe is located considerably anteriorly, but not at the anterior end (Fig. 3). In the opisthe, the anterior part is present in a very abbreviated form, and the anterior suture is very short and bent to the left. On the other hand, the opisthe has a much longer posterior part and longer posterior suture than those of a normal cell. It is notable that the cytoproct is also much longer than that of a normal cell (Fig. 3).

Other cells fail to complete cell division even after they are transferred into adenine-free fresh medium (Figs. 4, 5, 6). Since these cells are bent, they are referred to as L-shaped cells (Mishima, 1975). The L-shaped cell consists of an anterior member (AML) and a posterior member (PML). The L-shaped cell has two oral apparatuses which are located near the fission furrow (Figs. 4–7). The AML has a very long anterior suture and the PML has a long posterior suture with a long cytoproct (Fig. 4). Thus the ventral views of the AML and PML are similar to those of aberrantly shaped proters and opisthes, respectively.

Measurement of the length of the abnormally shaped cells indicates that these cells are nearly 90% as long as the normal interfission cells. Judging from silver-impregnated specimens, proliferation of kinetosomes and their arrangement in the preoral and postoral fields of the cell are not greatly affected. The presence of the long anterior suture and the posterior suture with long cytoproct may support this idea.

The aberrantly shaped proters, opisthes, and the L-shaped cells are collected separately and washed in 2 mM phosphate buffer (pH 7 ·2). They are then mixed with normally shaped cells of the opposite mating type (testers). Mating reactions take place between abnormally shaped cells and testers. The proters and the L-shaped cells sometimes adhere to two or more tester cells, whereas the opisthe adhere to only one. In normal cells fixed at 60 min after the beginning of the mating reaction, deciliated surfaces are observed along the preoral suture from the anterior end to near the buccal opening, and along the posterior right region of the buccal vestibulum (Fig. 8). The deciliated anterior portion participates in formation of the holdfast union pair. Holdfast union pairs are sometimes observed between a proter and a tester (Fig. 9); no such pairs are observed between an opisthe and a tester. In the aberrant proter, degeneration of cilia takes placs along the preoral sutures, which are very long, and in the cortical field to the right and posterior to the buccal vestibulum which is located near the posterior end of the cell (Fig. 10). In the aberrant opisthe, ciliary resorption takes place along the anterior suture, which is very short and bent to the left (Fig. 11), and in the posterior right field to the buccal vestibulum which is located considerably anteriorly (Fig. 11). Features of the deciliation in the L-shaped cells are very similar to those in the proters and the opisthes. Degeneration of cilia occurs along the long preoral suture of the AML (Fig. 12). The posterior right field, including the buccal vestibulum of PML, also becomes deciliated (Figs. 13–15). Conjugating pairs are sometimes formed between AML and the tester (Fig. 13). Thus the results show that the degeneration of cilia during conjugation takes place along almost the full length of the preoral suture, regardless of its total length, and at the posterior right of the buccal vestibulum, regardless of its location in the cell.

Although the mechanism of adenine action is still unknown (Mishima, 1975), similar anomalies have been induced with other chemicals such as phenethyl alcohol (Wille, 1966) and actinomycin D (Kaczanowska, Hyvert & de Haller, 1976). These works suggest that there are some common morphogenetic processes which are sensitive to these chemicals. In the adenine-induced anomalies, formation of the kinetosomes seems not to be greatly inhibited, but migration of the oral apparatus is severely restrained. As a result of this, newly formed kinetosomes are arranged into a preoral cortical pattern in the proter and AML, and postoral pattern in the opisthe and PML. According to Sonne-born (1977), the amount of cortical ciliary unit reproduction and the kinds of units produced are correlated with and, directly or indirectly, determined by the distance and direction from the oral area. Observation on the adenine-induced abnormally shaped cells supports this idea that the arrangement of new units is determined by their position relative to the oral area.

The present results confirm that the resorption of cilia takes place along the full length of the preoral suture, irrespective of its total length, and also in the region to the right and posterior to the buccal vestibule, irrespective of the position of the latter in the cell. In proters and AMLs ciliary degeneration takes place along the entire length of the abnormally long anterior suture. In opisthes and PMLs it takes place for about the normal distance along the abnormally long posterior suture. Thus there is evidence for spatial proportionality in the anterior cortex; but for absolute distance assessment in the posterior cortex.

It is known that the posterior right portion of the buccal vestibulum plays some important roles in formation of the new oral apparatus during sexual and asexual reproduction (Hanson, 1962; Hanson, Gillies & Kaneda, 1969; Kaneda & Hanson, 1974; Jones, 1976; Sonneborn, 1977). The paraoral cone, through which exchange of pronuclei takes place, is formed in this region during conjugation (Vivier & Andre, 1961 ; Hanson et al. 1969). Moreover, in some ami-cronucleate strains of P. caudatum, the region to the right and posterior to the buccal vestibulum in the vegetative cell (Fujishima & Watanabe, 1981) and exconjugants (Mikami, 1979) is without cilia. Thus the posterior right region is morphogenetically active judging from the high frequency of ciliary resorption observed there. The resorption in this region, however, may occur secondarily, because it begins first at the anterior suture and can be arrested before it extends to the buccal vestibulum region (Watanabe, 1978,1981).

The importance of the anterior suture in conjugation has also been pointed out in Tetrahymena (Wolfe & Grimes, 1979). Wolfe & Grimes (1979) reported that surface transformation takes place at the suture region before pairing to provide a site for joining the cells of complementary mating types. It is known that the suture is formed at the border between two different cortical fields (Sonneborn, 1963). The cytoproct, an organelle for excretion, is located on the posterior suture of Paramecium. Allen & Wolf (1974) clearly demonstrated that the movement of food vacuoles is oriented by cytoskeletal components towards the cytoproct. No such exocytotic excretion, however, has so far been observed at the anterior suture. On the other hand. Allen (1978 a, b) found, using freezefracture techniques, large rectangular plates of particles on the plasma membrane almost exclusively in the region of the anterior suture. Although the function of such membrane particle plates is still unknown, the location of the plates in the region of the anterior suture may have some relation, directly or indirectly, to the initiation and/or location of the degeneration of cilia during conjugation.

I should like to thank Drs G. de Haller, R. K. Peck and K. Hiwatashi for helpful discussions of this work, and for help in the preparation of the manuscript. This work was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan and by Research Grant No. 3.140.77 from the National Swiss Foundtion for Scientific Research to Drs G. de Haller and R. K. Peck.