J. Embryol. exp. Morph. Vol. 70, pp. 19-28, 1982
Printed in Great Britain © Company of Biologists Limited 1982
19
Correlation between ventral surface structures
and local degeneration of cilia during
conjugation in Paramecium
ByTSUYOSHI WATANABE1
From the Biological Institute, Tohoku University
and
Department of Animal Biology, University of Geneva
SUMMARY
Correlation between cortical structures, such as the suture lines and the oral apparatus,
and local degeneration of cilia during conjugation was investigated using aberrantly shaped
cells which were produced by treating normal cells with 2 mM adenine for 24 h. Three types
of aberrantly shaped cells were obtained: (1) proters, in which the oral apparatus is located
at or near the posterior end of the cell and the preoral suture is much longer than that of a
normally shaped cell; (2) opisthes, in which the oral apparatus is located extremely anteriorly,
the preoral suture is very short and bent to the left, and the postoral suture and cytoproct
are much longer than those of normally shaped cells; and (3) L-shaped cells, which are cells
that have not separated at division and contain two buccal openings. The anterior member
of the L-shaped cell (AML) has a long preoral suture and the posterior member of the Lshaped cell (PML) has a long postoral suture and cytoproct. The postoral field of AML
and the preoral field of PML are missing. When these cells were mixed with normally shaped
cells of the opposite mating type, the mating reaction took place between them. Scanning
electron microscopic observation showed that, in all abnormally shaped cells, the degeneration of cilia always occurred along the full length of the preoral suture, regardless of its total
length, and along the posterior right field of the buccal vestibulum, regardless of its location
in the cell cortex. The localization of the degeneration of cilia, therefore, coincided with the
position of the preoral suture and the buccal opening.
INTRODUCTION
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; ChenShan, 1969,1970; Kaneda & Hanson, 1974), In these works, the highly ordered
1
Author's address: Biological Institute, Tohoku University, Aoba-Yama, Sendai 980, Japan.
20
T. WATANABE
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 Ca 2+ 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).
MATERIALS AND METHODS
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-
Cortex o/Paramecium and local deciliation
21
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). Silverimpregnated 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.
RESULTS
Anomalies induced with adenine
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 raM 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-
22
T. WATANABE
AML
Cortex o/Paramecium and local deciliation
23
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.
Degeneration of cilia in the abnormally shaped cells
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,
Fig. 1. Silver preparation of normal cell. BO: buccal opening; AS: anterior suture;
PS: posterior suture; CP: cytoproct. x 550.
Fig. 2. Abnormally shaped proter. The oral apparatus is located at the posterior end
of the cell, x 550.
Fig. 3. Abnormally shaped opisthe. Buccal opening is located anteriorly. The anterior
suture is short and the posterior suture and cytoproct are long, x 550.
Fig. 4. L-shaped cell. AML and PML are connected by a cytoplasmic bridge, x 550.
Fig. 5. L-shaped cell. AML and PML are connected by a wide cytoplasmic bridge.
x300.
Fig. 6. L-shaped cell. Division furrow is poorly developed, x 300.
Fig. 7. Scanning electron micrograph of L-shaped cell, x 670.
24
T. WATANABE
Fig. 8. Normal cell 60 min after onset of mating reaction. Deciliation has occurred
along the anterior suture and in the region to the right and posterior to the buccal
opening. x800.
Fig. 9. Conjugating pair of an abnormally shaped proter (left) and a normally shaped
cell (right). x470.
Fig. 10. Enlargement of the posterior part of the conjugating proter in Fig. 9,90 min
after beginning of mating reaction. Ventral surface and the region to the right and
posterior to the buccal opening are deciliated. x 1000.
Fig. 11. The opisthe 90 min after onset of the mating reaction. Deciliation has occurred along the anterior suture (arrowhead) and in the region to the right and posterior
to the buccal opening, x 800.
regardless of its total length, and at the posterior right of the buccal vestibulum,
regardless of its location in the cell.
DISCUSSION
Adenine-induced abnormally shaped cells
Although the mechanism of adenine action is still unknown (Mishima, 1975),
similar anomalies have been induced with other chemicals such as phenethyl
Cortex o/Paramecium and local deciliation
Fig. 12. L-shaped cell. Ciliary resorption has taken place along the anterior suture
(arrowhead) of AML and in the region to the right and posterior to the buccal
opening (arrow) of PML. x 1000.
Fig. 13. Conjugating pair of a normal cell (N) and an L-shaped cell (L), 90 min after
the beginning of the mating reaction, x 330.
Fig. 14. Enlargement of the posterior part of the conjugating L-shaped cell in Fig. 13.
The region to the right and posterior to the buccal vestibulum is deciliated. x 1000.
Fig. 15. Middle portion of the L-shaped cell 60 min after the onset of the mating
reaction. The region to the right and posterior to the buccal vestibulum of the
PML is deciliated. x 1000.
25
26
T. WATANABE
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 Sonneborn (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.
Correlation between ciliary resorption and surface structure
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 amicronucleate 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
Cortex of Paramecium and local deciliaiion
27
the cytoproct. No such exocytotic excretion, however, has so far been observed
at the anterior suture. On the other hand. Allen (1978a, 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.
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(Received 27 November 1981, revised 1 February 1982)