J. Cell Sci. 3 a, S5-66 (1978)
Printed in Great Britain © Company of Biologists Limited 197S
55
A SCANNING ELECTRON-MICROSCOPIC
STUDY OF THE LOCAL DEGENERATION
OF CILIA DURING SEXUAL REPRODUCTION
IN PARAMECIUM
TSUYOSHI WATANABE
Biological Institute, Tohoku University, Sendai 980, Japan
SUMMARY
The location and extent of local degeneration of cilia during sexual reproduction of Paramecium was studied using scanning electron microscopy to examine cells undergoing conjugation and autogamy. At some time during the mating reaction, but prior to conjugant
pair formation, ciliary degeneration begins at the antero-ventral tip of cells and proceed*
posteriorly along the suture. In the anterior part of the cell, degeneration occurs on both sides
of the suture, but in the posterior part it is restricted to the right side of the suture. In 5 species
of Paramedum examined, degeneration occurred in nearly the same region. No degeneration
of cilia is observed in natural autogamy of P. tetranrelia, whereas in chemically induced autogamy of P. caiidatum degeneration occurs as in ordinary conjugation. Conjugant pairs never
expose any deciliated cell surface except at the postero-ventral tip. The maximum extent of
ciliary degeneration is best seen in the chemically induced autogamous cells: 7 kineties (rows
of unit teritories) at the anterior-left, 4 kineties at the anterior-right, 10 or more kineties at the
posterior-right and the right wall of the vestibule of the mouth. Before complete disappearance
of the cilia, many short cilia are observed. This suggests that ciliary degeneration is due to
resorption. Degeneration extends more rapidly in cells with stronger mating reactivity. The
relations between mating reactivity, ciliary degeneration and nuclear activation are discussed.
INTRODUCTION
In Paramedum and other ciliates, interesting studies of inheritance and morphogenesis of cortical pattern have been possible because of local differentiation in
structure and function of the cell surface (Sonneborn, 1963, 1970a). One notable
case is the set of events in conjugation restricted to the ventral surface of the cell.
In the conjugation process of Paramedum, cell contact is established in 3 steps:
mating reaction, holdfast union, and paroral union. In the mating reaction, cells of
complementary mating type stick together by 'mating reactive' cilia located on the
ventral surface of the cell (Hiwatashi, 1961). As the mating reaction proceeds, cilia
and trichocysts at the anterior tip and on the ventral surface just behind the tip
disappear, and pairs of cells unite at the antero-ventral surface in a 'holdfast union'.
Cilia continue to disappear on the ventral surface, and the cells unite more firmly,
especially in a region just posterior to the mouth, in a 'paroral union'. Details of the
holdfast and paroral unions may be found in Wichterman (1946), Hiwatashi (19556),
Vivier & Andre" (1961) and Miyake (1966). Hiwatashi (1961) suggested that there is
some relation between the contact region of the mating reaction and that of the
56
T. Watanabe
holdfast union. Moreover, the works of Hiwatashi (19556) and Miyake (1966,
1968 a, b) strongly indicate that the loss of cilia during conjugation is essential not
only for the formation of holdfast and paroral union but also for nuclear activation.
Thus the loss of cilia is an important phenomenon, not only from the viewpoint
of morphogenetic problems but also from that of fertilization (Miyake, 1974). However, the exact details and controlling mechanisms of the degeneration of cilia have
never been reported. To clarify these points, scanning electron-microscopic observations were made on cells undergoing conjugation and autogamy.
MATERIALS AND METHODS
Animals and culture methods
Stocks Kt, dKKi4a, 27aG3, dNi4a and di2-3~4 of Paramecium caudatum, syngen 3, were
used throughout the study. For conjugation via mating type, the cells were cultured with
lettuce juice medium (Hiwatashi, 1968) in which 1 vol. of fresh lettuce juice was diluted with
40 vol. of Dryl's solution (Dryl, 1959) and bacterized with Klebsiella aerogenes. For chemical
induction of autogamy, the cells were cultured with a Ca-poor medium in which lettuce juice
was diluted in 2 i m sodium phosphate buffer (pH 7-0) instead of Dryl's solution. For comparison, conjugating cells of P. tetraurelia (stock 51), P. multitnicronucleatum (CH-312 and
CH-313,) P. bwsaria (TK-i and TK-3) and P. trichium (PC-2 and PC-5) were used. Cells of
P. tetraurelia, stock 51, were also used for studies of natural autogamy.
Conjugation via mating type
Mating-reactive cells of complementary mating types were mixed in depression slides. The
cells agglutinated soon after mixing, and holdfast pairs were formed about 60 min after the
beginning of the mating reaction. Although the exact time of the onset of paroral union was
uncertain, firmly united pairs which had undergone paroral union were present after 90 min.
Such pairs were identified when drawing them into and expelling them from a pipette failed
to separate the 2 cells. Cells of P. caudatum were fixed at various times from 15 min to 5 h after
onset of the mating reaction. The cells of other Paramecium species were fixed at the time of
holdfast pair formation.
Strength of mating reactivity was assessed by noting if many clumps are formed quickly
after mixing mating types ('strong' or 'high' reactivity) or if few cells form clumps soon after
mixing ('weak' or 'low' reactivity). No method is currently available for determining the
amount of mating substance on each cell.
Chemical induction of autogamy
Chemical induction of autogamy was performed by a modification (Tsukii, in preparation)
of the method of Miyake (1968 a, b). The cells of a single mating type of P. caudatum were
cultured in Ca-poor medium and washed once in a modified Miyake's (1958) physiological
balanced solution called K-PBS-II (i-smMNaCl, r 8 mM KC1, o-irriMMgCl,, o-oi mM
CaClj, 18 mM KH2PO4 and 0-2 mM K,HPO4, pH 60). Then the cells were treated with the
autogamy-inducing medium (6 mM KC1, 50 mM methyl urea, and 40-80 /tg/ml ficin or 5-10
/*g/ml papain in K-PBS-II). Partially purified ficin was prepared from crude ficin (Wako
Pure Chemicals Co., Ltd.) according to the method of Hammond & Gutfreund (1959). Papain
used was a crystalline preparation (Sigma, 2 x crystallized). When the cells were treated with
the autogamy-inducing medium, neither agglutination nor pair formation was observed. At
20 h after the beginning of induction of autogamy, the occurrence of autogamy was ascertained
by looking for macronuclear fragmentation, a characteristic of sexual reproduction. Details of
the chemical induction of autogamy in P. caudatum will be described elsewhere (Tsukii, in
preparation).
Ciliary degeneration in Paramecium
m
Natural autogamy
Natural autogamy was induced in P. tetraurelia by starving sufficiently old cells (Sonneborn,
19706) at 27 CC. It is difficult to induce autogamy synchronously or to distinguish cells undergoing autogamy by external appearance. Therefore, to obtain a population of cells in various
stages of autogamy including early stages, cells were fixed when 10 % of the cells showed
macronuclear fragmentation. In unfixed controls, the proportion of cells with fragmented
macronuclei rose to 50 % 7 h after this time of fixation. In conjugation of P. aurelia, separation
of conjugants and macronuclear fragmentation occur 6-7 h after the initiation of conjugation
(Jurand & Selman, 1969).
Scanning electron microscopy
Cells were fixed in Parducz solution (Parducz, 1967) for 30 min at room temperature.
After washing in deionized water, the cells were dehydrated in a series of ethanol and isoamyl
acetate. The cells in isoamyl acetate were put on coverglasses or aluminium disks and airdried. The specimens were coated with gold and examined with an Hitach-Akashi MSM-4
scanning electron microscope.
Fig. 1. Silver preparation of P. caudatum (ventral view), al, anterior left field; ar,
anterior right field; as, anterior suture; pi, posterior left field; pr, posterior right
field; ps, posterior suture; v, vestibule, x 480.
T. Watanabe
Ciliary degeneration in Paramecium
59
RESULTS
Morphology of vegetative cells
The cortical structure of Paramecium has been described by many investigators
using the silver impregnation technique. The ventral morphology of the vegetative
cell will be described briefly. The most characteristic organelle of the ventral surface
is the mouth. The position of the opening of the mouth is somewhat different from
species to species. In P. caudatum, the mouth is located slightly posterior to the
centre of the cell. The suture runs longitudinally across the mouth. Therefore, the
ventral surface of Paramecium is divided into 4 parts by the suture and the mouth:
anterior-right, anterior-left, posterior-right and posterior-left (Figs. 1, 2). The
anterior-left corresponds to the oral groove, which extends from the anterior end to
the vestibule of the mouth and is apparent in living cells.
Order and location of ciliary degeneration in early stages of the conjugating process
The location of degenerating cilia and the order of their disappearance was examined
in cells of P. caudatum undergoing conjugation after the mating reaction. At 15 min
after the beginning of the mating reaction, no ciliary degeneration was observed.
At 30 min, when cells are still in the mating clumps, small numbers of cells show
short cilia or no cilia at their anterior tips (Fig. 3). Ciliary degeneration proceeds
posteriorly along the suture at about the time of holdfast pair formation. At this time,
some cells have short cilia of various lengths (Figs. 4, 5). This suggests that the
degeneration of cilia is due to resorption. The degeneration of cilia extends to the
sides and to the posterior end during the time of formation of holdfast to paroral
unions (Figs. 6, 7). At the posterior part, loss of cilia begins near the posterior-right
of the vestibule of the mouth (Fig. 8), and spreads to the posterior tip before formation of tight paroral union (Fig. 9). At the anterior part of the cell, the cilia were
lost on both sides of the anterior suture. On the contrary, at the posterior part,
ciliary degeneration was mainly restricted to the right side of the suture (Figs. 6-9).
The locations of ciliary degeneration during the early process of conjugation via
mating type were essentially identical in all species of Paramecium examined (Figs.
10-13). ^ n chemical induction of autogamy, which will be described later, the ciliary
degeneration occurred in the same order and at the same location as in the usual
conjugation. Thus the order and location of ciliary degeneration during conjugation is
Figs. 2-9. Scanning electron micrographs of ventral surface of P. caudatum. a,
anterior pole; /, left side; p, posterior pole; r, right side.
Fig. 2. Vegetative cell, x 850.
Fig. 3. 30 min after onset of mating reaction. Cell shows no cilia at anterior tip.
x8io.
Fig. 4. 60 min after onset of mating reaction. Note the short cilia of various lengths
along the anterior suture, x 2000.
Fig. 5. 80 min after onset of mating reaction. Degeneration of cilia extends to
both sides of the anterior suture, x 3300.
Iff
T. Watanabe
Ciliary degeneration in Paramecium
61
strikingly similar among species and among methods of induction. However, the
rate of degeneration of cilia was different from sample to sample. Generally, if the
cells have strong mating reactivity (see Methods), ciliary degeneration spreads more
rapidly. As a result, when mating reactivity is strong the formation of normal pairs is
accompanied by the formation of aberrant holdfast unions consisting of more than 3
cells (Fig. 14).
Extent of ciliary degeneration in later conjugation
The extent of ciliary degeneration was examined in firmly united conjugation pairs.
At 3 h, when pairs are united firmly, no bald free surface was observed except for
the last cell of multiple unions (Fig. 15), or for ventro-posterior tip of normal pairs
(Figs. 16, 17). No loss of cilia was observed on the dorsal surfaces and antero-ventral
part of any pair (Fig. 16), except for a few pairs which were found after a strong
mating reaction (Figs. 18, 19). These observations indicate that ciliary degeneration
occurs only on the ventral surface, including the region where the cells make contact.
For further study of the extent of ciliary degeneration, autogamous cells were examined, because in these there is no cell contact to hinder observation. Observations
were made on more than 100 cells of P. tetraurelia which were presumed to contain
more than 50 natural autogamous cells. Ciliary degeneration was not observed on
any cell. Since natural autogamy does not occur in other species of Paramecium,
cells of P. caudatum undergoing chemically induced autogamy were next studied.
Surprisingly, cells underwent ciliary degeneration. In order to exclude the possibility
that the chemicals induced the degeneration of cilia irrespective of mating ability of
the cell, immature cells, which had no ability to mate, were exposed to the same
induction medium. Neither ciliary degeneration nor macronuclear fragmentation
occurred. When the mating reactive cells were treated with ficin (40-80 /tg/ml) or
papain (5-10 /ig/ml) without KC1, no loss of cilia was detected. Thus ciliary loss is
not due to direct enzymic action by the proteolytic enzymes.
In Figs. 20-22, presumed autogamous cells obtained by chemical induction are
shown. The extent of ciliary degeneration 1-5 h after the onset of the treatment was
the same as that of usual conjugation at 1-5 h (Fig. 20). Ciliary degeneration reached
its maximum extent 3 h after the begninning of the induction of autogamy, because
no significant additional degeneration was observed after 5 h (Figs. 21, 22). Anterior
Fig. 6. 90 min after onset of mating reaction. Short cilia are still observed in the
anterior region, x 800.
Fig. 7. 90 min after onset of mating reaction. Degeneration of cilia has occurred on
both sides of the anterior suture and on the right side of the posterior suture, x 800.
Fig. 8. 60 min after onset of mating reaction. Early stage of ciliary loss in the posterior
portion of the cell, x 2000.
Fig. 9. 90 min after onset of mating reaction. Ciliary degeneration spreads to the
posterior tip before formation of tight paroral union, x 1300.
Fig. 10. P. tetraurelia. 80 min after onset of mating reaction, x 930.
Fig. 11. P. multimicronucleatum. 60 min after onset of mating reaction, x 400.
3
CKL
32
T. Watanabe
Ciliary degeneration in Paramecium
63
to the vestibule, the area of ciliary loss extended 7 kineties to the left of the suture
and 4 kineties to the right; posterior to the vestibule, it extended only 10 or more
kineties to the right (Figs. 23-25). The right wall of the vestibule was also deciliated
(Fig. 24).
DISCUSSION
The localization of ciliary degeneration during sexual reproduction is an interesting
morphogenetic problem in Paramecium. Mechanisms which might control ciliary
degeneration at a definite place and time have not yet been clarified. However, there
should be some relation between acquisition of mating reactivity and the degeneration of cilia, because ciliary degeneration occurs only in cells physiologically competent to give a mating reaction, and in cells with higher mating reactivity, ciliary
degeneration extends more rapidly. The facts that cells lose the capacity to mate
before undergoing natural autogamy and that they also show no degeneration of
cilia during autogamy also support the above hypothesis. Significantly, P. tetraurelia
does undergo ciliary degeneration when autogamy is induced chemically (T. M.
Sonneborn, personal communication).
Mating reactive cilia are distributed on the ventral surface of the cell (Hiwatashi,
1961; Cohen & Siegel, 1963; Cohen, 1964). In P. caudatum, if mating reactivity is
weak, cells contact mainly at the anterior portion, but if it is strong, they contact
over a much wider area (Hiwatashi, 1961). This suggests that in P. caudatum, the
strength of mating reactivity extends from anterior to posterior cilia. The pattern of
ciliary degeneration follows that of mating reactivity. This correlation may not be
significant, since in P. bursaria mating reactivity spreads from posterior to anterior
(Cohen, 1964), whereas we have observed ciliary degeneration extending from
anterior to posterior. Therefore, there is no direct relation between the pattern of
development of mating reactivity and of ciliary degeneration in P. bursaria, though
the final locations of both phenomena seem essentially the same.
The location of ciliary degeneration is identical in all 5 species of Paramecium
examined in this study: both sides of the anterior suture and the right side of the
posterior suture. Vivier & Andre" (1961) cut serial sections of conjugant pairs and
Fig. 12. P. bursaria. 150 min after onset of mating reaction, x 930.
Fig. 13. P. trichium. 150 min after onset of mating reaction, x 1300.
Fig. 14. Aberrant multiple union of P. caudatum (90 min). x 160.
Fig. 15. Enlargement of Fig. 14. Deciliated surface is apparent (arrow), x 400.
Fig. 16. Normal paroral union, 3 h after onset of mating reaction. No deciliated
surface is observed except for the posterior end (arrow), x 530.
Fig. 17. The same sample as Fig. 16, but it is rotated about 90° and enlarged. Both
cells show deciliated surface on the posterior portion (arrows). X 1300.
Fig. 18. An aberrant pair which is rarely observed after strong mating reaction. The
lower cell shows bald free surface at the anterior end. x 400.
Fig. 19. Enlargement of the anterior portion of Fig. 18. Bald free surface is apparent
(arrow), x
1200.
5-2
T. Watanabe
Ciliary degeneration in Paramecium
65
showed that cells unite at the right side of the suture. However, it seems likely that
cells may unite on both sides of the anterior suture, because ciliary degeneration
occurs on both sides of it and one never sees on conjugating cells any exposed deciliated surface on the anterior part of the cell. In our experience, it is difficult to determine the location of the suture with certainty in sectioned material.
Others have proposed that the loss of cilia during conjugation is due to their
separation from the cell body (Vivier & Andre", 1961; Bloodgood, 1974). However,
the present observations of many short cilia during degeneration suggest that cilia
may be lost by resorption. Since the electron-microscopic observations on late
conjugating pairs show that kinetosomes remain intact at the contact region (Vivier &
Andre", 1961; Jurand & Selman, 1969; T. Watanabe, unpublished), degeneration of
each cilium seems to stop at the region between the kinetosome and the ciliary shaft.
Resorption of cilia has been reported in other ciliates: during conjugation of Oxytricha (Hammersmith, 1976) and during oral replacement and partial deciliation in
Tetrahymena (Williams & Nelsen, 1973; Rannestad, 1974). As shown in Tetrahymena,
active resorption mechanism(s) act at a specific position and a specific time in the
cell cycle (Williams, 1975). Likewise in the conjugation of Paramecium, resorption
mechanism(s) act at a specific position, the ventral side of the cell, and at a specific
time, namely, when cells are activated by the ciliary mating reaction or by chemical
treatment inducing conjugation or autogamy.
The extent of ciliary degeneration reached its maximum 3 h after beginning of the
chemical induction of autogamy. Miyake (19686) reported that if the cells of P.
multimicronuckatum were treated with autogamy-inducing chemicals for 3 h, nuclear
activation was induced. Similarly, in P. caudatum, the minimum length of the chemical
treatment necessary for nuclear activation is 3 h (Y. Tsukii, unpublished). Hiwatashi
(1955a) reported that paroral union, which is formed more than 2 h after the start of
the mating reaction, is essential for nuclear activation in the conjugation of P. caudatum. These results suggest that the time for nuclear activation corresponds closely
to that of the maximum extension of ciliary degeneration. Studies are still in progress
to discover if there is a causal role for ciliary degeneration in cell contact and nuclear
activation in Paramecium.
Figs. 20-25. Cells of P. caudatum which were treated with autogamy-inducing
medium.
Fig. 20. 1-5 h after onset of the treatment, x 1000.
Fig. 21. 3 h after onset of the treatment, x 1000.
Fig. 22. 5 h after onset of the treatment, x 600.
Fig. 23. Anterior portion of a cell treated with autogamy-inducing medium for 3 h.
At the right side of the suture, deciliation has reached 4 kineties. At the left side of the
suture, ciliary degeneration is in progress. Small arrows indicate the kineties or the
unit teritories where cilia have disappeared, x 3300.
Fig. 24. Enlargement of the middle portion of Fig. 22. Note deciliation in right
wall of vestibule, x 2000.
Fig. 25. The same sample as Fig. 24 but rotated about 900. The left wall of vestibule has not deciliated. x sooo.
66
T. Watanahe
The author is much indebted to D. L. Cronkite for his help in the preparation of the manuscript, and would like to thank Dr T. M. Sonneborn and Dr K. Hiwatashi for reading the
manuscript, and Dr K. Mikami and Mr Y. Tsukii for technical assistance. This work was
supported by a grant from the Ministry of Education of Japan.
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