J. Cell Sci. 3a, S5-66 (1978) 55Printed in Great Britain © Company of Biologists Limited 197S

A SCANNING ELECTRON-MICROSCOPIC

STUDY OF THE LOCAL DEGENERATION

OF CILIA DURING SEXUAL REPRODUCTION

IN PARAMECIUM

TSUYOSHI WATANABEBiological Institute, Tohoku University, Sendai 980, Japan

SUMMARY

The location and extent of local degeneration of cilia during sexual reproduction of Para-mecium was studied using scanning electron microscopy to examine cells undergoing con-jugation and autogamy. At some time during the mating reaction, but prior to conjugantpair 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 sidesof the suture, but in the posterior part it is restricted to the right side of the suture. In 5 speciesof Paramedum examined, degeneration occurred in nearly the same region. No degenerationof cilia is observed in natural autogamy of P. tetranrelia, whereas in chemically induced auto-gamy of P. caiidatum degeneration occurs as in ordinary conjugation. Conjugant pairs neverexpose any deciliated cell surface except at the postero-ventral tip. The maximum extent ofciliary degeneration is best seen in the chemically induced autogamous cells: 7 kineties (rowsof unit teritories) at the anterior-left, 4 kineties at the anterior-right, 10 or more kineties at theposterior-right and the right wall of the vestibule of the mouth. Before complete disappearanceof the cilia, many short cilia are observed. This suggests that ciliary degeneration is due toresorption. Degeneration extends more rapidly in cells with stronger mating reactivity. Therelations between mating reactivity, ciliary degeneration and nuclear activation are discussed.

INTRODUCTION

In Paramedum and other ciliates, interesting studies of inheritance and morpho-genesis of cortical pattern have been possible because of local differentiation instructure and function of the cell surface (Sonneborn, 1963, 1970a). One notablecase 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 ofcomplementary mating type stick together by 'mating reactive' cilia located on theventral surface of the cell (Hiwatashi, 1961). As the mating reaction proceeds, ciliaand trichocysts at the anterior tip and on the ventral surface just behind the tipdisappear, 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 theholdfast and paroral unions may be found in Wichterman (1946), Hiwatashi (19556),Vivier & Andre" (1961) and Miyake (1966). Hiwatashi (1961) suggested that there issome 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 notonly 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 viewpointof morphogenetic problems but also from that of fertilization (Miyake, 1974). How-ever, the exact details and controlling mechanisms of the degeneration of cilia havenever been reported. To clarify these points, scanning electron-microscopic obser-vations 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, wereused throughout the study. For conjugation via mating type, the cells were cultured withlettuce juice medium (Hiwatashi, 1968) in which 1 vol. of fresh lettuce juice was diluted with40 vol. of Dryl's solution (Dryl, 1959) and bacterized with Klebsiella aerogenes. For chemicalinduction of autogamy, the cells were cultured with a Ca-poor medium in which lettuce juicewas diluted in 2 i m sodium phosphate buffer (pH 7-0) instead of Dryl's solution. For com-parison, conjugating cells of P. tetraurelia (stock 51), P. multitnicronucleatum (CH-312 andCH-313,) P. bwsaria (TK-i and TK-3) and P. trichium (PC-2 and PC-5) were used. Cells ofP. 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. Thecells agglutinated soon after mixing, and holdfast pairs were formed about 60 min after thebeginning of the mating reaction. Although the exact time of the onset of paroral union wasuncertain, 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 failedto separate the 2 cells. Cells of P. caudatum were fixed at various times from 15 min to 5 h afteronset of the mating reaction. The cells of other Paramecium species were fixed at the time ofholdfast pair formation.

Strength of mating reactivity was assessed by noting if many clumps are formed quicklyafter mixing mating types ('strong' or 'high' reactivity) or if few cells form clumps soon aftermixing ('weak' or 'low' reactivity). No method is currently available for determining theamount 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 werecultured in Ca-poor medium and washed once in a modified Miyake's (1958) physiologicalbalanced solution called K-PBS-II (i-smMNaCl, r 8 mM KC1, o-irriMMgCl,, o-oi mMCaClj, 18 mM KH2PO4 and 0-2 mM K,HPO4, pH 60). Then the cells were treated with theautogamy-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 (WakoPure Chemicals Co., Ltd.) according to the method of Hammond & Gutfreund (1959). Papainused was a crystalline preparation (Sigma, 2 x crystallized). When the cells were treated withthe autogamy-inducing medium, neither agglutination nor pair formation was observed. At20 h after the beginning of induction of autogamy, the occurrence of autogamy was ascertainedby looking for macronuclear fragmentation, a characteristic of sexual reproduction. Details ofthe chemical induction of autogamy in P. caudatum will be described elsewhere (Tsukii, inpreparation).

Ciliary degeneration in Paramecium mNatural 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 under-going autogamy by external appearance. Therefore, to obtain a population of cells in variousstages of autogamy including early stages, cells were fixed when 10 % of the cells showedmacronuclear fragmentation. In unfixed controls, the proportion of cells with fragmentedmacronuclei rose to 50 % 7 h after this time of fixation. In conjugation of P. aurelia, separationof 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 isoamylacetate. The cells in isoamyl acetate were put on coverglasses or aluminium disks and air-dried. The specimens were coated with gold and examined with an Hitach-Akashi MSM-4scanning 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 rightfield; ps, posterior suture; v, vestibule, x 480.

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Ciliary degeneration in Paramecium 59

RESULTS

Morphology of vegetative cells

The cortical structure of Paramecium has been described by many investigatorsusing the silver impregnation technique. The ventral morphology of the vegetativecell will be described briefly. The most characteristic organelle of the ventral surfaceis the mouth. The position of the opening of the mouth is somewhat different fromspecies to species. In P. caudatum, the mouth is located slightly posterior to thecentre of the cell. The suture runs longitudinally across the mouth. Therefore, theventral 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). Theanterior-left corresponds to the oral groove, which extends from the anterior end tothe 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 examinedin cells of P. caudatum undergoing conjugation after the mating reaction. At 15 minafter 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 showshort cilia or no cilia at their anterior tips (Fig. 3). Ciliary degeneration proceedsposteriorly 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 thedegeneration of cilia is due to resorption. The degeneration of cilia extends to thesides and to the posterior end during the time of formation of holdfast to paroralunions (Figs. 6, 7). At the posterior part, loss of cilia begins near the posterior-rightof the vestibule of the mouth (Fig. 8), and spreads to the posterior tip before for-mation of tight paroral union (Fig. 9). At the anterior part of the cell, the cilia werelost 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 viamating type were essentially identical in all species of Paramecium examined (Figs.10-13). ^n chemical induction of autogamy, which will be described later, the ciliarydegeneration occurred in the same order and at the same location as in the usualconjugation. 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.

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Ciliary degeneration in Paramecium 61

strikingly similar among species and among methods of induction. However, therate of degeneration of cilia was different from sample to sample. Generally, if thecells have strong mating reactivity (see Methods), ciliary degeneration spreads morerapidly. As a result, when mating reactivity is strong the formation of normal pairs isaccompanied by the formation of aberrant holdfast unions consisting of more than 3cells (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 forthe 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-ventralpart of any pair (Fig. 16), except for a few pairs which were found after a strongmating reaction (Figs. 18, 19). These observations indicate that ciliary degenerationoccurs 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 ex-amined, because in these there is no cell contact to hinder observation. Observationswere made on more than 100 cells of P. tetraurelia which were presumed to containmore than 50 natural autogamous cells. Ciliary degeneration was not observed onany 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 possibilitythat the chemicals induced the degeneration of cilia irrespective of mating ability ofthe cell, immature cells, which had no ability to mate, were exposed to the sameinduction medium. Neither ciliary degeneration nor macronuclear fragmentationoccurred. When the mating reactive cells were treated with ficin (40-80 /tg/ml) orpapain (5-10 /ig/ml) without KC1, no loss of cilia was detected. Thus ciliary loss isnot due to direct enzymic action by the proteolytic enzymes.

In Figs. 20-22, presumed autogamous cells obtained by chemical induction areshown. The extent of ciliary degeneration 1-5 h after the onset of the treatment wasthe same as that of usual conjugation at 1-5 h (Fig. 20). Ciliary degeneration reachedits maximum extent 3 h after the begninning of the induction of autogamy, becauseno 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 theanterior region, x 800.

Fig. 7. 90 min after onset of mating reaction. Degeneration of cilia has occurred onboth 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 posteriorportion of the cell, x 2000.

Fig. 9. 90 min after onset of mating reaction. Ciliary degeneration spreads to theposterior 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 sutureand 4 kineties to the right; posterior to the vestibule, it extended only 10 or morekineties 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 interestingmorphogenetic problem in Paramecium. Mechanisms which might control ciliarydegeneration at a definite place and time have not yet been clarified. However, thereshould be some relation between acquisition of mating reactivity and the degener-ation of cilia, because ciliary degeneration occurs only in cells physiologically com-petent to give a mating reaction, and in cells with higher mating reactivity, ciliarydegeneration extends more rapidly. The facts that cells lose the capacity to matebefore undergoing natural autogamy and that they also show no degeneration ofcilia during autogamy also support the above hypothesis. Significantly, P. tetraureliadoes 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 isweak, cells contact mainly at the anterior portion, but if it is strong, they contactover a much wider area (Hiwatashi, 1961). This suggests that in P. caudatum, thestrength of mating reactivity extends from anterior to posterior cilia. The pattern ofciliary degeneration follows that of mating reactivity. This correlation may not besignificant, since in P. bursaria mating reactivity spreads from posterior to anterior(Cohen, 1964), whereas we have observed ciliary degeneration extending fromanterior to posterior. Therefore, there is no direct relation between the pattern ofdevelopment of mating reactivity and of ciliary degeneration in P. bursaria, thoughthe final locations of both phenomena seem essentially the same.

The location of ciliary degeneration is identical in all 5 species of Parameciumexamined in this study: both sides of the anterior suture and the right side of theposterior 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 deciliatedsurface 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. Bothcells show deciliated surface on the posterior portion (arrows). X 1300.Fig. 18. An aberrant pair which is rarely observed after strong mating reaction. Thelower 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

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Ciliary degeneration in Paramecium 65

showed that cells unite at the right side of the suture. However, it seems likely thatcells may unite on both sides of the anterior suture, because ciliary degenerationoccurs on both sides of it and one never sees on conjugating cells any exposed decili-ated surface on the anterior part of the cell. In our experience, it is difficult to deter-mine the location of the suture with certainty in sectioned material.

Others have proposed that the loss of cilia during conjugation is due to theirseparation from the cell body (Vivier & Andre", 1961; Bloodgood, 1974). However,the present observations of many short cilia during degeneration suggest that ciliamay be lost by resorption. Since the electron-microscopic observations on lateconjugating pairs show that kinetosomes remain intact at the contact region (Vivier &Andre", 1961; Jurand & Selman, 1969; T. Watanabe, unpublished), degeneration ofeach 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 Oxy-tricha (Hammersmith, 1976) and during oral replacement and partial deciliation inTetrahymena (Williams & Nelsen, 1973; Rannestad, 1974). As shown in Tetrahymena,active resorption mechanism(s) act at a specific position and a specific time in thecell cycle (Williams, 1975). Likewise in the conjugation of Paramecium, resorptionmechanism(s) act at a specific position, the ventral side of the cell, and at a specifictime, namely, when cells are activated by the ciliary mating reaction or by chemicaltreatment inducing conjugation or autogamy.

The extent of ciliary degeneration reached its maximum 3 h after beginning of thechemical induction of autogamy. Miyake (19686) reported that if the cells of P.multimicronuckatum were treated with autogamy-inducing chemicals for 3 h, nuclearactivation was induced. Similarly, in P. caudatum, the minimum length of the chemicaltreatment 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 ofthe mating reaction, is essential for nuclear activation in the conjugation of P. cauda-tum. These results suggest that the time for nuclear activation corresponds closelyto that of the maximum extension of ciliary degeneration. Studies are still in progressto discover if there is a causal role for ciliary degeneration in cell contact and nuclearactivation in Paramecium.

Figs. 20-25. Cells of P. caudatum which were treated with autogamy-inducingmedium.

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 thesuture, ciliary degeneration is in progress. Small arrows indicate the kineties or theunit teritories where cilia have disappeared, x 3300.

Fig. 24. Enlargement of the middle portion of Fig. 22. Note deciliation in rightwall of vestibule, x 2000.

Fig. 25. The same sample as Fig. 24 but rotated about 900. The left wall of vesti-bule 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 manu-script, and would like to thank Dr T. M. Sonneborn and Dr K. Hiwatashi for reading themanuscript, and Dr K. Mikami and Mr Y. Tsukii for technical assistance. This work wassupported by a grant from the Ministry of Education of Japan.

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{Received 22 August 1977 - Revised 6 January 1978)