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Defensive function of trichocysts in Paramecium against the predatory ciliate Monodinium balbiani
Europ. J. Protisto!' 32, 128-133 (1996) February 23, 1996
European Journal of
PROTISTOLOGY
Defensive Function of Trichocysts in Paramecium against the Predatory Ciliate Monodinium balbiani Akio Miyake and Terue Harumoto* Dipartimento di Bi%gia Mo/eco/are Cellu/are ed Anima/e, UniversitiJ. di Camerino, 62032 Camerino (MC), Italy
SUMMARY Defensive function of trichocysts in Paramecium was recently verified against the predatory ciliate Dileptus margaritifer. Until then, however, their defensive function had been repeatedly questioned based on the observation that trichocysts are of little use against Didinium nasutum. We tested whether trichocysts can defend Paramecium against Monodinium, a predatory ciliate closely related to Didinium. Cells of trichocyst-non-discharge (tnd) mutants, artificially induced trichocyst-deficient cells, and intact wild-type cells of Paramecium were compared as prey for M. balbiani. In both P. caudatum and P. tetraurelia, mutant cells were much more vulnerable than wild-type cells (killed 12-40 times more often by the predator under a certain condition). Cells of P. tetraurelia with reduced numbers of trichocysts, obtained by treating wild-type cells with lysozyme, were almost as vulnerable as mutant cells. On the contrary, when tested against D. nasutum, wild-type and mutant cells of P. tetraurelia were equally vulnerable. We conclude that discharge of trichocysts defends Paramecium against M. balbiani but not against D. nasutum. These results strongly suggest that trichocysts in Paramecium function as defensive organelles against many toxicyst-bearing predatory ciliates with a few exceptions such as D. nasutum.
Introduction Defensive function of trichocysts in Paramecium was recently verified by Harumoto and Miyake [5, Abstr. 8th Internat. Congr. Protozoo!. Tsukuba 1989, p. 76] who showed that discharge of trichocysts defends Paramecium against the predatory ciliate Dileptus margaritifer. This was confirmed by Knoll, Haacke-Bell and Plattner [9]. Harumoto [4] also showed that the role of avoiding reaction is minor, if any, compared to trichocyst discharge in the defense of Paramecium
"Present address: Department of Biology, Faculty of Science, Nara Women's University, 630 Nara, Japan 0932-4739-96-0032-0128$3.50-0
against D. margaritifer. These results strongly support the century-old hypothesis [1, 7, 12] that trichocysts in Paramecium are organelles of defense. In the past, however, this hypothesis had been repeatedly questioned. Indeed the dispute about the defensive function of trichocysts was once dominated by the negative evidence that trichocyst discharge of Paramecium is of little use against Didinium nasutum [3, 8, 17,23,28,29]. Any work on the defensive function of trichocysts must take into account this fact. We, therefore, examined whether trichocysts can defend Paramecium against Monodinium balbiani, a predatory ciliate closely related to D. nasutum. In this work we show that trichocysts in Paramecium are effective in the defense against M. balbiani. We also confirm the earlier observations that these organelles are not effective in the defense against D. nasutum. © 1996 by Gustav Fischer Verlag, Stuttgart
Defensive Function of Trichocysts . 129
Based on these results we propose that the defense by trichocysts in Paramecium should be effective against many toxicyst-bearing predatory ciliates and that the failure in defense against Didinium is an exceptional case. Material and Methods Cells. Stock M7 of Monodinium balbiani, collected from a pond in Munster, Germany in 1993 (Fig. 1), Stock 7771 of Didinium nasutum, one of the stocks derived from the ATCC strain 30399 purchased from American Type Culture Collection, Stocks Kyk402 and 27aG3 of Paramecium caudatum, and Stocks 51 [21], d4-2 [21], and nd7 of Paramecium tetraurelia were used. Stocks 27aG3 [25] and nd7 [101 are trichocyst-nan-discharge (tnd) mutants; they do not discharge trichocysts when exposed to the stimulus which induces the discharge of trichocysts in wild-type cells. P. caudatum and P. tetraurelia were grown and suspended in 5MB-III [14] (described as 5MB below) as in 15]. M. balbiani was grown on an 5MB suspension of Saprophilus sp. which was grown in the same way as Paramecium. D. nasutum was grown on an 5MB suspension of P. tetraurelia, stock 51 unless otherwise indicated. All cells used for experiments were moderately starved cells in the stationary phase. Preparation of trichocyst-deficient cells. Cells of P. tetraurelia stock 51 were treated by 100 ~lg/ml egg-white lysozyme (Boehringer Mannheim) in 5MB for 30 sec, washed with 5MB, and used after 2 h. The treatment induced a massive discharge of trichocysts in most cells without seriously injuring them [5].
Index of trichocyst-discharging capacity (tdc index). The capacity of trichocyst discharge in Paramecium was estimated by measuring the tdc index [5]. A suspension of Paramecium was mixed with the same volume of saturated solution of picric acid (a strong inducer of trichocyst discharge) and the extent of trichocyst discharge of individual cells was recorded as one of the four classes, 1) full discharge, 2) intermediate discharge, 3) discharge of up to three trichocysts, and 4) no discharge. The tdc index of a population is a series of percentages of cells in these four classes of trichocyst discharge that were observed in 100 cells (e.g., the index is 0-0-0-100 if all cells do not discharge any trichocysts). Lugol's solution. An aqueous solution of potassium iodide (2.5%) and iodine (1.25%) was prepared as the quarter concentrated Lugol's solution (114 Lugol). This or a further diluted solution (1/10-1120 Lugol) was dropped on a 50100 III suspension of M. balbiani, P. tetraurelia or M. balbiani + P. tetraurelia placed on a glass slide. M. balbiani was killed discharging toxicysts to various extents (Fig. 1), while P. tetraurelia was killed scarcely discharging trichocysts. Culture, handling of cells, and experiments were performed at 23 ± 1 0c.
Results Trichocyst-non-discharge (tnd) mutants and wildtype cells of P. caudatum and P. tetraurelia were compared as prey for M. balbiani. Ten paramecia, mutant or wild-type, were placed in a 250 III 5MB suspension of monodinia (100-1000 cells/ml), and surviving paramecia were counted after 1 or 2 h. In both species of Paramecium, more cells disappeared in mutant than in wild-type cells at every monodinia density tested (Figs. 2 and 3): In P. tetraurelia (200 monodinia/ml) and P. caudatum (250 monodinia/ml), 12 and 40 times more mutant cells disappeared, respectively. Tnd mutants are, therefore, much more vulnerable than wild-type cells to M. balbiani. 10 8
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Fig. 1. Toxicyst-discharging Monodinium balbiani stock M7. Fixed and stained in 1110 Lugol's solution. Phase contrast micrograph, x 850.
Fig. 2. Predation by Monodinium balbiani on wild-type and tnd-mutant cells of Paramecium tetraurelia. The number of surviving paramecia after 2h is plotted against monodinia density. 0 - - 0 : wild-type cells (stock 51).0-0: wild-type cells (stock d4-2). 6 - - 6 : tnd-mutant cells (stock nd7). Mean and SD of three experiments.
130 . A. Miyake and T. Harumoto 100--0-. 8
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Fig. 3. Predation by Monodinium balbiani on wild-type and tnd-mutant cells of Paramecium caudatum. The number of surviving paramecia after 1 h is plotted against monodinia density. 0 - 0 : wild-type cells (stock Kyk402). tc.-tc.: tnd-mutant cells (stock 27aG3). Mean and SD ofthree experiments.
Wild-type and tnd-mutant cells of Paramecium were compared also by observing individual cells. A single cell of Paramecium was placed in a 250 III 5MB suspension of M. balbiani (1600 cells/ml for P. tetraurelia, 1000 cells/ml for P. caudatum) and the Paramecium cell was followed under a stereomicroscope. For wild-type cells of P. tetraurelia, stock 51 was used. A successful hunter of M. balbiani first stuck to a prey ("catching") and immediately started to engulf it. The observation was discontinued when the Paramecium cell was caught or when 5 min had elapsed after the beginning of the observation. The observation was repeated 20 times. In P. tetraurelia, mutant cells were caught in 47.4 ± 48.9 sec (mean ± SD, N = 20), all within 5 min, while not a single wild-type cell was caught in 5 min. In P. caudatum, mutant cells were caught in 51.1 ± 64.0 sec (mean ± SD, N = 20), all within 5 min, while only one wild-type cell was caught in 5 min (at 4.5 min). The result again shows that tnd-mutants are much more vulnerable to M. balbiani than wild-type cells. In these observations, mutant cells were usually caught at the first hit by the predator. On the contrary, wild-type cells usually escaped from the hit by rapidly swimming away, indicating that the observed difference is due to the capacity of wild-type cells to ward off the predator rather than the capacity to avoid an encounter with the predator. Although wild-type cells are caught much less frequently, once caught, they are almost invariably engulfed just as mutant cells. The immunity of wildtype cells against M. balbiani is, therefore, due to their ability to avoid the catching or the firm sticking to the predator. Since the main difference between tnd-mutants and wild-type paramecia are the lack of the capacity to dis-
charge trichocysts in the mutant, it is likely that the observed immunity of wild-type cells against M. balbiani is due to this capacity. The following two experiments were carried out to test this hypothesis. In the first experiment, wild-type cells of P. tetraurelia (stock 51) were mixed with monodinia on a glass slide. After 30 sec, when no paramecia had yet been caught, cells were fixed and stained by 1/4 Lugol and observed with a phase-contrast microscope. Many discharged trichocysts were scattered in the field. Since trichocyst discharge was scarcely induced when only Paramecium was fixed by 1/4 Lugol, the result indicates that Paramecium discharges trichocysts when encountering M. balbiani. In the second experiment, artificially induced trichocyst deficient cells, intact control cells (both stock 51) and tnd-mutant cells of P. tetraurelia were compared as prey for M. balbiani. The trichocyst-deficient cells were produced by treating stock 51 cells by lysozyme as described in Material and Methods. Control cells of stock 51 and tnd-mutant cells were treated with 5MB instead of the lysozyme solution. The trichocyst-discharging capacity of lysozyme-treated cells expressed by tdc index was 0-35-27-38, while the indices for control cells of stock 51 and tnd-mutant cells were 96-4-0-0 and 00-0-100, respectively. Ten paramecia were placed in a 250 III 5MB suspension of monodinia and surviving paramecia were counted after 2 h. More cells disappeared in lysozyme-treated cells than in control cells at every monodinia density tested. Slightly more cells disappeared in mutant cells than in lysozyme-treated wild-type cells (Fig. 4). The vulnerability of Paramecium to M. balbiani is, therefore, inversely related to the capacity of trichocyst discharge. 10 8
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Fig. 4. Predation by Monodinium balbiani on cells of Paramecium tetraurelia having different capacities of trichocyst
discharge. The number of surviving paramecia after 2 h is plotted against monodinia density. 0 - 0 : intact wild-type cells (stock 51).•- . : artificially induced trichocyst deficient cells (stock 51). tc.-tc.: tnd-mutant cells (stock nd7). Tdc indices of the intact wild-type cells, the trichocyst deficient cells, and mutant cells were 96-4-0-0, 0-35-27-38, and 0-0-0-100, respectively, at the time the experiment started. Mean and SD of three experiments.
Defensive Function of Trichocysts . 131
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Fig. 5. Predation by Didinium nasutum on wild-type (stock 51) and tnd-mutant (stock nd?) cells of Paramecium tetraurelia. D. nasutum had been fed for 1 week before the experiment on stock 51 (A) or nd? (B) of P. tetraurelia. The number of surviving paramecia after 1 h is plotted against the cell density of didinia. 0 - 0 : wild-type cells (stock 51). !::.-!::.: tnd-mutant cells (stock nd?). Mean and SD of three experiments.
Both of these results are consistent with the hypothesis. We conclude that trichocyst discharge can defend Paramecium against M. balbiani. We also compared wild-type cells and tnd-mutant cells of P. tetraurelia as prey against D. nasutum using the same method as in the experiments of Figs. 2-4. Wild-type and mutant cells were equally vulnerable to D. nasutum, as shown in earlier works [3, 17], irrespective of whether Didinium had been previously fed on wild-type cells or mutant cells of Paramecium (Fig. SA-B). This confirms earlier observations that trichocyst discharge in Paramecium is of little use against D. nasutum [3,8, 17,23,28,29]. Trichocyst discharge is, therefore, effective in defending Paramecium against M. balbiani but not against D. nasutum under the same experimental conditions.
Discussion A defensive function of trichocysts in Paramecium has been verified against Dileptus margaritifer [5,9, 15] and refuted against Didinium nasutum [3, 8, 17,23,28,29]. The present work, which verifies the defensive function of trichocysts against Monodinium balbiani, not only shows that the defensive function of trichocysts is by no means limited against D. margaritifer, but also enables to estimate the value of trichocysts as defensive organelles as discussed below. D. margaritifer and M. balbiani are considerably different predators. They are classified in different orders of the class Litostomatea [20]. Although both use toxicysts in the proboscis to capture prey, they capture and engulf it in quite different ways. D. margaritifer paralyzes or disintegrates a prey by hitting it with the long flexible toxicyst-bearing proboscis and takes the prey with the mouth situated at the base of the proboscis. M. balbiani sticks to its prey with the short coneshaped toxicyst-bearing proboscis, paralyzes the prey, and engulfs it through the proboscis which also functions as a mouth. The successful defense against both of them strongly suggests that trichocysts are effective against various toxicyst-bearing predatory ciliates. On the other hand, D. nasutum and M. balbiani are similar predators. They are both classified in the family Didiniidae [20] and are quite similar in cortical ultrastructural organization [18]. They are also alike in hunting behavior. Both species stick to a prey with the cone-shaped proboscis, paralyze the prey and then start to engulf it. The successful defense against M. balbiani strongly suggests that the ineffectiveness of trichocysts against D. nasutum is an exceptional case. It is, therefore, likely that trichocysts in Paramecium function as organelles of defense against many toxicystbearing predatory ciliates. The heavy investment by Paramecium to carry thousands of these fairly large extrusomes is then understandable, even if they have no other function. Many functions other than defense have been proposed for trichocysts in Paramecium. They include cortical strengthening [2], attachment to substratum [19], osmoregulation [30], role in conjugation [24] and communication [A. Miyake, personal communication cited in 1]. According to Haacke-Bell and collaborators [3], who critically examined many of these and other proposed functions of trichocysts, the only available clear evidence for any of the examined possible functions was the experimental demonstration of the defensive function against Dileptus [S cited as in press]. They concluded that trichocysts in Paramecium probably function as defensive organelles, although several other possible functions deserve study. These studies suggest that trichocysts in other ciliates and flagellates are also organelles for defense against predators. The function of trichocysts in flagellates is unknown, but possible functions proposed for them include defense, propulsive movement by local discharge, and feeding [22]. Studies on trichocysts in Paramecium
132 . A. Miyake and T. Harumoto
support not only the first but also the second hypothesis, because the propulsive movement by local discharge of trichocysts is assumed to be an important defense mechanism of these organelles [5,9]. The present work enables to probe into the problem, what makes D. nasutum a successful hunter of Paramecium, by examining differences between D. nasutum and M. balbiani. The most prominent diagnostic difference between these two predators is in the number of ciliary girdles; two in D. nasutum, one in M. balbiani. This is, however, unlikely to be a decisive factor, if any, for the successful hunting of Paramecium, because the two species look very alike in swimming behavior. Rodrigues de Santa Rosa and Didier [18], who studied the ultrastructure of M. balbiani, note three differences between D. nasutum and M. balbiani. First, they found only one type of extrusomes, toxicysts, in the proboscis of M. balbiani, while in the proboscis of D. nasutum, two types of extrusomes, pexicysts and toxicysts, had been found [26]. Second, a fibrous ring at the base of the proboscis described in D. nasutum [26] is lacking in M. balbiani. Third, cyrtocysts, toxicyst-like extrusomes of unknown function described in D. nasutum [26], were not found in M. balbiani. Cyrtocysts are distributed all over the body surface of D. nasutum except at the proboscis region [26]. Since the successful hunting of Paramecium by D. nasutum depends on the firm attachment to the prey with the proboscis, cyrtocysts, which are absent in the proboscis, are unlikely to be essential for Paramecium hunting. The function of the fibrous ring is unknown and is not considered further here. However, the reported difference in extrusomes in the proboscis deserves discussion. Wessenberg and Antipa [27], who describe how D. nasutum hunts P. multimicronucleatum, assume that the discharge of pexicysts is the first response of prey recognition, and this discharge in turn induces the discharge of toxicysts. When the Didinium proboscis hits a Paramecium cell, the latter discharges many trichocysts, mostly from the hit side. The impact of this trichocyst discharge separates the two cells, but the proboscis is still joined with the prey by a slender connection which they interpret as a bundle of discharged pexicysts and toxicysts of Didinium. The Paramecium cell stops swimming, the connection shortens, and the cell is engulfed by the predator. If pexicysts in D. nasutum participate in prey catching, and if the observation by Rodrigues de Santa Rosa and Didier [18] is interpreted to indicate a lack of pexicysts in M. balbiani, success versus failure in hunting Paramecium by D. nasutum and M. balbiani can be ascribed to the presence or absence of pexicysts. If this is the case, one may assume that D. nasutum has overcome the defense mechanism of Paramecium by acquiring pexicysts. Unfortunately, however, this hypothesis must be taken with reservation. According to Wessenberg and Antipa [26,27], toxicysts are several times longer than pexicysts, but they are similar in ultrastructure. Santa
Rosa and Didier, whose observation was on sectioned materials for electron microscopy, note that their toxicysts are of different dimensions [18]. They, therefore, cannot rule out the possibility that M. balbiani has pexicysts. In our preliminary observations on crushed cells of M. balbiani, we observed two types of extrusomelike structures both apparently derived from the proboscis. One of them is 3-4 times longer than the other. If they are toxicysts and pexicysts, respectively, the above hypothesis is untenable. As an alternative, D: nasutum might have overcome the defense mechanism of Paramecium by increasing the number of pexicysts and toxicysts and/or by improving their efficiency, for example, by making the discharge of toxicysts quicker. If Paramecium responds to the first stage of the attack (pexicyst discharge) by discharging trichocysts before the second, more lethal stage of the attack (toxicyst discharge), the cell-cell connection made of only pexicysts may be torn off by the impact of trichocyst discharge. To postulate such a quick response is not unreasonable, since it has been shown that Paramecium can discharge trichocysts within 1 millisecond in response to electrical triggering [11, 13], as pointed out in [5]. Based on the fact that some extrusomes, such as toxicysts, haptocysts, pexicysts, function as offensive organelles and that some other extrusomes, such as trichocysts in Paramecium and pigment granules in Blepharisma, function as defensive organelles, Miyake and collaborators [16] proposed a unifying hypothesis that extrusomes in free-living protists are organelles primarily for the offense-defense interaction. The result of this work and the recent finding that pigment granules in Stentor coeruleus also function as defensive organelles against D. margaritifer [6] are consistent with this hypothesis.
Acknowledgements We thank Drs. J. Beisson and J. Cohen of Centre National de la Recherche Scientifique, Gif-sur-Yvette, France, for providing a tnd mutant (nd?) of P. tetraurelia; Dr. Y. Tsukii of Hosei University, Tokyo, for providing a tnd mutant (27aG3) of P. caudatum; Dr. M. Takahashi, University ofTsukuba, for providing a wild-type stock (Kyk402) of P. caudatum; Drs. K. Heckmann and H.-W. Kuhlmann, Munster University, Munster, for the help in collecting M. balbiani; Ms. V. Rivola for technical assistance. This work was supported by Italian CNR.
References 1 Adoutte A. (1988): Exocytosis: biogenesis, transport and secretion of trichocysts. In: Gortz H.-D. (ed.) Paramecium, pp. 325 - 362. Springer-Verlag, Berlin. 2 Ehret C. D. and McArdle E. W. (1974): The structure of Paramecium as viewed from its constituent levels of organization. In: van Wagtendonk W. J. (ed.): Parame-
Defensive Function of Trichocysts . 133
3
4
5 6 7 8 9
10
11
12 13 14
15
16
cium - A current survey, pp. 263-338. Elsevier, Amsterdam. Haacke-Bell B., Hohenberger-Bregger R., and Plattner H. (1990): Trichocysts of Paramecium: secretory organelles in search of their function. Europ. J. Protisto!', 25, 289-305. Harumoto T. (1994): The role of trichocyst discharge and backward swimming in escaping behavior of Paramecium from Dileptus margaritifer. J. Euk. Microbio!., 41, 560564. Harumoto T and Miyake A. (1991): Defensive function of trichocysts in Paramecium. J. Exp. Zoo!., 260, 84-92. Harumoto T and Miyake A. (1994): Defensive function of pigment granules in Stentor. Zoo!' Sci., 11 Supp!., 24. Hausmann K. (1978): Extrusive organelles in protists. Int. Rev. Cyto!., 52,197-276. Jurand A. and Selman G. G. (1969): The anatomy of Paramecium aurelia. Macmillan St. Martin's Press, New York. Knoll G., Haacke-Bell B., and Plattner H. (1991): Local trichocyst exocytosis provides an efficient escape mechanism for Paramecium cells. Europ. J. Protisto!', 27,381385. Lefort-Tran M. K., Aufderheide M., Pouphile M., Rossignol M., and Beisson J. (1981): Control of exocytotic processes: cytological and physiological studies of trichocyst mutants in Paramecium tetraurelia. J. Cell Bio!., 88, 301-311. Matt H., Bilinski M., and Plattner H. (1978): Adenosine triphosphate, calcium and temperature requirements for the final steps of exocytosis in Paramecium cells. J. Cell Sci., 32, 67 - 86. Maupas E. (1883): Contribution a letude morphologique et anatomique des infusioires cilies. Arch. Zoo!' Exp. Gen., 1,427-664. Millwe D. M., Jahn T L., and Fonseca J. R. (1968): Anodal contraction of Paramecium protoplasm. J. Protozoo!., 15,493-497. Miyake A. (1981): Cell interaction by gamones in Blepharisma. In: O'Day D. H. and Horgen P. A. (eds.): Sexual reproduction in eukaryotic microbes, pp. 95-129. Academic Press, New York. Miyake A., Harumoto T, Salvi B., and Rivola V. (1989): Defensive functions of extrusomes, pigment granules in Blepharisma and trichocysts in Paramecium, against a carnivorous ciliate Dileptus. J. Protozoo!., 36, 28A. Miyake A., Harumoto T., Salvi B., and Rivola V. (1990): Defensive function of pigment granules in Blepharisma japonicum. Europ. J. Protisto!', 25, 310 - 315.
17 Pollack S. (1974): Mutations affecting the trichocysts in Paramecium aurelia. 1. Morphology and description of the mutants. J. Protozoo!., 21, 352 - 362. 18 Rodrigues de Santa Rosa M. et Didier P. (1975): Remarques sur l'ultrastructure du cilie gymnostome Monodinium balbiani (Fabre Domergue, 1888). Protistologica, 11, 469-479. 19 Saunders J. T (1925): The trichocysts of Paramecium. Philos. Trans. R. Soc. Lond. [Ser. Bj, 1,249-269. 20 Small E. B. and Lynn D. H. (1985): Phylum Ciliophora Doflein, 1901. In: Lee J. J., Hutner S. H., and Bovee E. C. (eds.): An illustrated guide to the protozoa, pp. 393-575. Soc. Protozoo!., Lawrence, Kansas. 21 Sonneborn T M. (1974): Paramecium aurelia. In: King R. C. (ed.): Handbook of genetics 2, pp. 469-594. Plenum Press, New York. 22 Vickerman K., Brugerolle G., and Mignot J.-P. (1991): Mastigophora. In: Harrison F. W. and Corliss J. O. (eds.): Microscopic anatomy of invertebrates, vo!. 1: Protozoa, pp. 13-159. Wiley-Liss, New York. 23 Vivier E. (1974): Morphology, taxonomy and general biology of the genus Paramecium. In: van Wagtendonk W. J. (ed.) : Paramecium - a current survey, pp. 1- 89. Elsevier, Amsterdam. 24 Vivier E. et Andre J. (1961): Donnees structurales et ultrastructurales nouvelles sur la conjugaison de Paramecium caudatum. J. Protozoo!., 8, 416-426. 25 Watanabe T and Takei K. (1982): Exocytosis defective mutants in Paramecium caudatum. Zoo!' Magazine, 91,580. 26 Wessenberg H. and Antipa G. (1968): Studies on Didinium nasutum. I. Structure and ultrastructure. Protistologica, 4, 427-447. 27 Wessenberg H. and Antipa G. (1970): Capture and ingestion of Paramecium by Didinium nasutum. J. Protozoo!., 17,250-270. 28 Wichterman R. (1953): The biology of Paramecium. Blakiston, New York. 29 Wichterman R. (1986): The biology of Paramecium. 2nd cd. Plenum Press, New York. 30 Wohlfarth-Bottermann K. E. (1953): Experimentelle und elektronenoptische Untersuchungen zur Funktion der Trichocysten von Paramecium caudatum. Arch. Protistenk., 98, 169-226.
Key words: Trichocysts - Predator-prey interaction - Paramecium - Monodinium - Didinium Akio Miyake, Dipartimento di Biologia MCA, Universita di Camerino, Via F. Camerini 2, 62032 Camerino (MC), Italy
European Journal of
PROTISTOLOGY
Defensive Function of Trichocysts in Paramecium against the Predatory Ciliate Monodinium balbiani Akio Miyake and Terue Harumoto* Dipartimento di Bi%gia Mo/eco/are Cellu/are ed Anima/e, UniversitiJ. di Camerino, 62032 Camerino (MC), Italy
SUMMARY Defensive function of trichocysts in Paramecium was recently verified against the predatory ciliate Dileptus margaritifer. Until then, however, their defensive function had been repeatedly questioned based on the observation that trichocysts are of little use against Didinium nasutum. We tested whether trichocysts can defend Paramecium against Monodinium, a predatory ciliate closely related to Didinium. Cells of trichocyst-non-discharge (tnd) mutants, artificially induced trichocyst-deficient cells, and intact wild-type cells of Paramecium were compared as prey for M. balbiani. In both P. caudatum and P. tetraurelia, mutant cells were much more vulnerable than wild-type cells (killed 12-40 times more often by the predator under a certain condition). Cells of P. tetraurelia with reduced numbers of trichocysts, obtained by treating wild-type cells with lysozyme, were almost as vulnerable as mutant cells. On the contrary, when tested against D. nasutum, wild-type and mutant cells of P. tetraurelia were equally vulnerable. We conclude that discharge of trichocysts defends Paramecium against M. balbiani but not against D. nasutum. These results strongly suggest that trichocysts in Paramecium function as defensive organelles against many toxicyst-bearing predatory ciliates with a few exceptions such as D. nasutum.
Introduction Defensive function of trichocysts in Paramecium was recently verified by Harumoto and Miyake [5, Abstr. 8th Internat. Congr. Protozoo!. Tsukuba 1989, p. 76] who showed that discharge of trichocysts defends Paramecium against the predatory ciliate Dileptus margaritifer. This was confirmed by Knoll, Haacke-Bell and Plattner [9]. Harumoto [4] also showed that the role of avoiding reaction is minor, if any, compared to trichocyst discharge in the defense of Paramecium
"Present address: Department of Biology, Faculty of Science, Nara Women's University, 630 Nara, Japan 0932-4739-96-0032-0128$3.50-0
against D. margaritifer. These results strongly support the century-old hypothesis [1, 7, 12] that trichocysts in Paramecium are organelles of defense. In the past, however, this hypothesis had been repeatedly questioned. Indeed the dispute about the defensive function of trichocysts was once dominated by the negative evidence that trichocyst discharge of Paramecium is of little use against Didinium nasutum [3, 8, 17,23,28,29]. Any work on the defensive function of trichocysts must take into account this fact. We, therefore, examined whether trichocysts can defend Paramecium against Monodinium balbiani, a predatory ciliate closely related to D. nasutum. In this work we show that trichocysts in Paramecium are effective in the defense against M. balbiani. We also confirm the earlier observations that these organelles are not effective in the defense against D. nasutum. © 1996 by Gustav Fischer Verlag, Stuttgart
Defensive Function of Trichocysts . 129
Based on these results we propose that the defense by trichocysts in Paramecium should be effective against many toxicyst-bearing predatory ciliates and that the failure in defense against Didinium is an exceptional case. Material and Methods Cells. Stock M7 of Monodinium balbiani, collected from a pond in Munster, Germany in 1993 (Fig. 1), Stock 7771 of Didinium nasutum, one of the stocks derived from the ATCC strain 30399 purchased from American Type Culture Collection, Stocks Kyk402 and 27aG3 of Paramecium caudatum, and Stocks 51 [21], d4-2 [21], and nd7 of Paramecium tetraurelia were used. Stocks 27aG3 [25] and nd7 [101 are trichocyst-nan-discharge (tnd) mutants; they do not discharge trichocysts when exposed to the stimulus which induces the discharge of trichocysts in wild-type cells. P. caudatum and P. tetraurelia were grown and suspended in 5MB-III [14] (described as 5MB below) as in 15]. M. balbiani was grown on an 5MB suspension of Saprophilus sp. which was grown in the same way as Paramecium. D. nasutum was grown on an 5MB suspension of P. tetraurelia, stock 51 unless otherwise indicated. All cells used for experiments were moderately starved cells in the stationary phase. Preparation of trichocyst-deficient cells. Cells of P. tetraurelia stock 51 were treated by 100 ~lg/ml egg-white lysozyme (Boehringer Mannheim) in 5MB for 30 sec, washed with 5MB, and used after 2 h. The treatment induced a massive discharge of trichocysts in most cells without seriously injuring them [5].
Index of trichocyst-discharging capacity (tdc index). The capacity of trichocyst discharge in Paramecium was estimated by measuring the tdc index [5]. A suspension of Paramecium was mixed with the same volume of saturated solution of picric acid (a strong inducer of trichocyst discharge) and the extent of trichocyst discharge of individual cells was recorded as one of the four classes, 1) full discharge, 2) intermediate discharge, 3) discharge of up to three trichocysts, and 4) no discharge. The tdc index of a population is a series of percentages of cells in these four classes of trichocyst discharge that were observed in 100 cells (e.g., the index is 0-0-0-100 if all cells do not discharge any trichocysts). Lugol's solution. An aqueous solution of potassium iodide (2.5%) and iodine (1.25%) was prepared as the quarter concentrated Lugol's solution (114 Lugol). This or a further diluted solution (1/10-1120 Lugol) was dropped on a 50100 III suspension of M. balbiani, P. tetraurelia or M. balbiani + P. tetraurelia placed on a glass slide. M. balbiani was killed discharging toxicysts to various extents (Fig. 1), while P. tetraurelia was killed scarcely discharging trichocysts. Culture, handling of cells, and experiments were performed at 23 ± 1 0c.
Results Trichocyst-non-discharge (tnd) mutants and wildtype cells of P. caudatum and P. tetraurelia were compared as prey for M. balbiani. Ten paramecia, mutant or wild-type, were placed in a 250 III 5MB suspension of monodinia (100-1000 cells/ml), and surviving paramecia were counted after 1 or 2 h. In both species of Paramecium, more cells disappeared in mutant than in wild-type cells at every monodinia density tested (Figs. 2 and 3): In P. tetraurelia (200 monodinia/ml) and P. caudatum (250 monodinia/ml), 12 and 40 times more mutant cells disappeared, respectively. Tnd mutants are, therefore, much more vulnerable than wild-type cells to M. balbiani. 10 8
.!!!
~ E
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4
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200
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400
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600
I
800
Monodinia (celis/mil
Fig. 1. Toxicyst-discharging Monodinium balbiani stock M7. Fixed and stained in 1110 Lugol's solution. Phase contrast micrograph, x 850.
Fig. 2. Predation by Monodinium balbiani on wild-type and tnd-mutant cells of Paramecium tetraurelia. The number of surviving paramecia after 2h is plotted against monodinia density. 0 - - 0 : wild-type cells (stock 51).0-0: wild-type cells (stock d4-2). 6 - - 6 : tnd-mutant cells (stock nd7). Mean and SD of three experiments.
130 . A. Miyake and T. Harumoto 100--0-. 8
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2
200
400
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Monodinia (cells/ml)
Fig. 3. Predation by Monodinium balbiani on wild-type and tnd-mutant cells of Paramecium caudatum. The number of surviving paramecia after 1 h is plotted against monodinia density. 0 - 0 : wild-type cells (stock Kyk402). tc.-tc.: tnd-mutant cells (stock 27aG3). Mean and SD ofthree experiments.
Wild-type and tnd-mutant cells of Paramecium were compared also by observing individual cells. A single cell of Paramecium was placed in a 250 III 5MB suspension of M. balbiani (1600 cells/ml for P. tetraurelia, 1000 cells/ml for P. caudatum) and the Paramecium cell was followed under a stereomicroscope. For wild-type cells of P. tetraurelia, stock 51 was used. A successful hunter of M. balbiani first stuck to a prey ("catching") and immediately started to engulf it. The observation was discontinued when the Paramecium cell was caught or when 5 min had elapsed after the beginning of the observation. The observation was repeated 20 times. In P. tetraurelia, mutant cells were caught in 47.4 ± 48.9 sec (mean ± SD, N = 20), all within 5 min, while not a single wild-type cell was caught in 5 min. In P. caudatum, mutant cells were caught in 51.1 ± 64.0 sec (mean ± SD, N = 20), all within 5 min, while only one wild-type cell was caught in 5 min (at 4.5 min). The result again shows that tnd-mutants are much more vulnerable to M. balbiani than wild-type cells. In these observations, mutant cells were usually caught at the first hit by the predator. On the contrary, wild-type cells usually escaped from the hit by rapidly swimming away, indicating that the observed difference is due to the capacity of wild-type cells to ward off the predator rather than the capacity to avoid an encounter with the predator. Although wild-type cells are caught much less frequently, once caught, they are almost invariably engulfed just as mutant cells. The immunity of wildtype cells against M. balbiani is, therefore, due to their ability to avoid the catching or the firm sticking to the predator. Since the main difference between tnd-mutants and wild-type paramecia are the lack of the capacity to dis-
charge trichocysts in the mutant, it is likely that the observed immunity of wild-type cells against M. balbiani is due to this capacity. The following two experiments were carried out to test this hypothesis. In the first experiment, wild-type cells of P. tetraurelia (stock 51) were mixed with monodinia on a glass slide. After 30 sec, when no paramecia had yet been caught, cells were fixed and stained by 1/4 Lugol and observed with a phase-contrast microscope. Many discharged trichocysts were scattered in the field. Since trichocyst discharge was scarcely induced when only Paramecium was fixed by 1/4 Lugol, the result indicates that Paramecium discharges trichocysts when encountering M. balbiani. In the second experiment, artificially induced trichocyst deficient cells, intact control cells (both stock 51) and tnd-mutant cells of P. tetraurelia were compared as prey for M. balbiani. The trichocyst-deficient cells were produced by treating stock 51 cells by lysozyme as described in Material and Methods. Control cells of stock 51 and tnd-mutant cells were treated with 5MB instead of the lysozyme solution. The trichocyst-discharging capacity of lysozyme-treated cells expressed by tdc index was 0-35-27-38, while the indices for control cells of stock 51 and tnd-mutant cells were 96-4-0-0 and 00-0-100, respectively. Ten paramecia were placed in a 250 III 5MB suspension of monodinia and surviving paramecia were counted after 2 h. More cells disappeared in lysozyme-treated cells than in control cells at every monodinia density tested. Slightly more cells disappeared in mutant cells than in lysozyme-treated wild-type cells (Fig. 4). The vulnerability of Paramecium to M. balbiani is, therefore, inversely related to the capacity of trichocyst discharge. 10 8
.~
6
Q)
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III
4
a.
2
200
400
600
800
Monodinia (cells/ml)
Fig. 4. Predation by Monodinium balbiani on cells of Paramecium tetraurelia having different capacities of trichocyst
discharge. The number of surviving paramecia after 2 h is plotted against monodinia density. 0 - 0 : intact wild-type cells (stock 51).•- . : artificially induced trichocyst deficient cells (stock 51). tc.-tc.: tnd-mutant cells (stock nd7). Tdc indices of the intact wild-type cells, the trichocyst deficient cells, and mutant cells were 96-4-0-0, 0-35-27-38, and 0-0-0-100, respectively, at the time the experiment started. Mean and SD of three experiments.
Defensive Function of Trichocysts . 131
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400
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Didinia (cells/ml)
Fig. 5. Predation by Didinium nasutum on wild-type (stock 51) and tnd-mutant (stock nd?) cells of Paramecium tetraurelia. D. nasutum had been fed for 1 week before the experiment on stock 51 (A) or nd? (B) of P. tetraurelia. The number of surviving paramecia after 1 h is plotted against the cell density of didinia. 0 - 0 : wild-type cells (stock 51). !::.-!::.: tnd-mutant cells (stock nd?). Mean and SD of three experiments.
Both of these results are consistent with the hypothesis. We conclude that trichocyst discharge can defend Paramecium against M. balbiani. We also compared wild-type cells and tnd-mutant cells of P. tetraurelia as prey against D. nasutum using the same method as in the experiments of Figs. 2-4. Wild-type and mutant cells were equally vulnerable to D. nasutum, as shown in earlier works [3, 17], irrespective of whether Didinium had been previously fed on wild-type cells or mutant cells of Paramecium (Fig. SA-B). This confirms earlier observations that trichocyst discharge in Paramecium is of little use against D. nasutum [3,8, 17,23,28,29]. Trichocyst discharge is, therefore, effective in defending Paramecium against M. balbiani but not against D. nasutum under the same experimental conditions.
Discussion A defensive function of trichocysts in Paramecium has been verified against Dileptus margaritifer [5,9, 15] and refuted against Didinium nasutum [3, 8, 17,23,28,29]. The present work, which verifies the defensive function of trichocysts against Monodinium balbiani, not only shows that the defensive function of trichocysts is by no means limited against D. margaritifer, but also enables to estimate the value of trichocysts as defensive organelles as discussed below. D. margaritifer and M. balbiani are considerably different predators. They are classified in different orders of the class Litostomatea [20]. Although both use toxicysts in the proboscis to capture prey, they capture and engulf it in quite different ways. D. margaritifer paralyzes or disintegrates a prey by hitting it with the long flexible toxicyst-bearing proboscis and takes the prey with the mouth situated at the base of the proboscis. M. balbiani sticks to its prey with the short coneshaped toxicyst-bearing proboscis, paralyzes the prey, and engulfs it through the proboscis which also functions as a mouth. The successful defense against both of them strongly suggests that trichocysts are effective against various toxicyst-bearing predatory ciliates. On the other hand, D. nasutum and M. balbiani are similar predators. They are both classified in the family Didiniidae [20] and are quite similar in cortical ultrastructural organization [18]. They are also alike in hunting behavior. Both species stick to a prey with the cone-shaped proboscis, paralyze the prey and then start to engulf it. The successful defense against M. balbiani strongly suggests that the ineffectiveness of trichocysts against D. nasutum is an exceptional case. It is, therefore, likely that trichocysts in Paramecium function as organelles of defense against many toxicystbearing predatory ciliates. The heavy investment by Paramecium to carry thousands of these fairly large extrusomes is then understandable, even if they have no other function. Many functions other than defense have been proposed for trichocysts in Paramecium. They include cortical strengthening [2], attachment to substratum [19], osmoregulation [30], role in conjugation [24] and communication [A. Miyake, personal communication cited in 1]. According to Haacke-Bell and collaborators [3], who critically examined many of these and other proposed functions of trichocysts, the only available clear evidence for any of the examined possible functions was the experimental demonstration of the defensive function against Dileptus [S cited as in press]. They concluded that trichocysts in Paramecium probably function as defensive organelles, although several other possible functions deserve study. These studies suggest that trichocysts in other ciliates and flagellates are also organelles for defense against predators. The function of trichocysts in flagellates is unknown, but possible functions proposed for them include defense, propulsive movement by local discharge, and feeding [22]. Studies on trichocysts in Paramecium
132 . A. Miyake and T. Harumoto
support not only the first but also the second hypothesis, because the propulsive movement by local discharge of trichocysts is assumed to be an important defense mechanism of these organelles [5,9]. The present work enables to probe into the problem, what makes D. nasutum a successful hunter of Paramecium, by examining differences between D. nasutum and M. balbiani. The most prominent diagnostic difference between these two predators is in the number of ciliary girdles; two in D. nasutum, one in M. balbiani. This is, however, unlikely to be a decisive factor, if any, for the successful hunting of Paramecium, because the two species look very alike in swimming behavior. Rodrigues de Santa Rosa and Didier [18], who studied the ultrastructure of M. balbiani, note three differences between D. nasutum and M. balbiani. First, they found only one type of extrusomes, toxicysts, in the proboscis of M. balbiani, while in the proboscis of D. nasutum, two types of extrusomes, pexicysts and toxicysts, had been found [26]. Second, a fibrous ring at the base of the proboscis described in D. nasutum [26] is lacking in M. balbiani. Third, cyrtocysts, toxicyst-like extrusomes of unknown function described in D. nasutum [26], were not found in M. balbiani. Cyrtocysts are distributed all over the body surface of D. nasutum except at the proboscis region [26]. Since the successful hunting of Paramecium by D. nasutum depends on the firm attachment to the prey with the proboscis, cyrtocysts, which are absent in the proboscis, are unlikely to be essential for Paramecium hunting. The function of the fibrous ring is unknown and is not considered further here. However, the reported difference in extrusomes in the proboscis deserves discussion. Wessenberg and Antipa [27], who describe how D. nasutum hunts P. multimicronucleatum, assume that the discharge of pexicysts is the first response of prey recognition, and this discharge in turn induces the discharge of toxicysts. When the Didinium proboscis hits a Paramecium cell, the latter discharges many trichocysts, mostly from the hit side. The impact of this trichocyst discharge separates the two cells, but the proboscis is still joined with the prey by a slender connection which they interpret as a bundle of discharged pexicysts and toxicysts of Didinium. The Paramecium cell stops swimming, the connection shortens, and the cell is engulfed by the predator. If pexicysts in D. nasutum participate in prey catching, and if the observation by Rodrigues de Santa Rosa and Didier [18] is interpreted to indicate a lack of pexicysts in M. balbiani, success versus failure in hunting Paramecium by D. nasutum and M. balbiani can be ascribed to the presence or absence of pexicysts. If this is the case, one may assume that D. nasutum has overcome the defense mechanism of Paramecium by acquiring pexicysts. Unfortunately, however, this hypothesis must be taken with reservation. According to Wessenberg and Antipa [26,27], toxicysts are several times longer than pexicysts, but they are similar in ultrastructure. Santa
Rosa and Didier, whose observation was on sectioned materials for electron microscopy, note that their toxicysts are of different dimensions [18]. They, therefore, cannot rule out the possibility that M. balbiani has pexicysts. In our preliminary observations on crushed cells of M. balbiani, we observed two types of extrusomelike structures both apparently derived from the proboscis. One of them is 3-4 times longer than the other. If they are toxicysts and pexicysts, respectively, the above hypothesis is untenable. As an alternative, D: nasutum might have overcome the defense mechanism of Paramecium by increasing the number of pexicysts and toxicysts and/or by improving their efficiency, for example, by making the discharge of toxicysts quicker. If Paramecium responds to the first stage of the attack (pexicyst discharge) by discharging trichocysts before the second, more lethal stage of the attack (toxicyst discharge), the cell-cell connection made of only pexicysts may be torn off by the impact of trichocyst discharge. To postulate such a quick response is not unreasonable, since it has been shown that Paramecium can discharge trichocysts within 1 millisecond in response to electrical triggering [11, 13], as pointed out in [5]. Based on the fact that some extrusomes, such as toxicysts, haptocysts, pexicysts, function as offensive organelles and that some other extrusomes, such as trichocysts in Paramecium and pigment granules in Blepharisma, function as defensive organelles, Miyake and collaborators [16] proposed a unifying hypothesis that extrusomes in free-living protists are organelles primarily for the offense-defense interaction. The result of this work and the recent finding that pigment granules in Stentor coeruleus also function as defensive organelles against D. margaritifer [6] are consistent with this hypothesis.
Acknowledgements We thank Drs. J. Beisson and J. Cohen of Centre National de la Recherche Scientifique, Gif-sur-Yvette, France, for providing a tnd mutant (nd?) of P. tetraurelia; Dr. Y. Tsukii of Hosei University, Tokyo, for providing a tnd mutant (27aG3) of P. caudatum; Dr. M. Takahashi, University ofTsukuba, for providing a wild-type stock (Kyk402) of P. caudatum; Drs. K. Heckmann and H.-W. Kuhlmann, Munster University, Munster, for the help in collecting M. balbiani; Ms. V. Rivola for technical assistance. This work was supported by Italian CNR.
References 1 Adoutte A. (1988): Exocytosis: biogenesis, transport and secretion of trichocysts. In: Gortz H.-D. (ed.) Paramecium, pp. 325 - 362. Springer-Verlag, Berlin. 2 Ehret C. D. and McArdle E. W. (1974): The structure of Paramecium as viewed from its constituent levels of organization. In: van Wagtendonk W. J. (ed.): Parame-
Defensive Function of Trichocysts . 133
3
4
5 6 7 8 9
10
11
12 13 14
15
16
cium - A current survey, pp. 263-338. Elsevier, Amsterdam. Haacke-Bell B., Hohenberger-Bregger R., and Plattner H. (1990): Trichocysts of Paramecium: secretory organelles in search of their function. Europ. J. Protisto!', 25, 289-305. Harumoto T. (1994): The role of trichocyst discharge and backward swimming in escaping behavior of Paramecium from Dileptus margaritifer. J. Euk. Microbio!., 41, 560564. Harumoto T and Miyake A. (1991): Defensive function of trichocysts in Paramecium. J. Exp. Zoo!., 260, 84-92. Harumoto T and Miyake A. (1994): Defensive function of pigment granules in Stentor. Zoo!' Sci., 11 Supp!., 24. Hausmann K. (1978): Extrusive organelles in protists. Int. Rev. Cyto!., 52,197-276. Jurand A. and Selman G. G. (1969): The anatomy of Paramecium aurelia. Macmillan St. Martin's Press, New York. Knoll G., Haacke-Bell B., and Plattner H. (1991): Local trichocyst exocytosis provides an efficient escape mechanism for Paramecium cells. Europ. J. Protisto!', 27,381385. Lefort-Tran M. K., Aufderheide M., Pouphile M., Rossignol M., and Beisson J. (1981): Control of exocytotic processes: cytological and physiological studies of trichocyst mutants in Paramecium tetraurelia. J. Cell Bio!., 88, 301-311. Matt H., Bilinski M., and Plattner H. (1978): Adenosine triphosphate, calcium and temperature requirements for the final steps of exocytosis in Paramecium cells. J. Cell Sci., 32, 67 - 86. Maupas E. (1883): Contribution a letude morphologique et anatomique des infusioires cilies. Arch. Zoo!' Exp. Gen., 1,427-664. Millwe D. M., Jahn T L., and Fonseca J. R. (1968): Anodal contraction of Paramecium protoplasm. J. Protozoo!., 15,493-497. Miyake A. (1981): Cell interaction by gamones in Blepharisma. In: O'Day D. H. and Horgen P. A. (eds.): Sexual reproduction in eukaryotic microbes, pp. 95-129. Academic Press, New York. Miyake A., Harumoto T, Salvi B., and Rivola V. (1989): Defensive functions of extrusomes, pigment granules in Blepharisma and trichocysts in Paramecium, against a carnivorous ciliate Dileptus. J. Protozoo!., 36, 28A. Miyake A., Harumoto T., Salvi B., and Rivola V. (1990): Defensive function of pigment granules in Blepharisma japonicum. Europ. J. Protisto!', 25, 310 - 315.
17 Pollack S. (1974): Mutations affecting the trichocysts in Paramecium aurelia. 1. Morphology and description of the mutants. J. Protozoo!., 21, 352 - 362. 18 Rodrigues de Santa Rosa M. et Didier P. (1975): Remarques sur l'ultrastructure du cilie gymnostome Monodinium balbiani (Fabre Domergue, 1888). Protistologica, 11, 469-479. 19 Saunders J. T (1925): The trichocysts of Paramecium. Philos. Trans. R. Soc. Lond. [Ser. Bj, 1,249-269. 20 Small E. B. and Lynn D. H. (1985): Phylum Ciliophora Doflein, 1901. In: Lee J. J., Hutner S. H., and Bovee E. C. (eds.): An illustrated guide to the protozoa, pp. 393-575. Soc. Protozoo!., Lawrence, Kansas. 21 Sonneborn T M. (1974): Paramecium aurelia. In: King R. C. (ed.): Handbook of genetics 2, pp. 469-594. Plenum Press, New York. 22 Vickerman K., Brugerolle G., and Mignot J.-P. (1991): Mastigophora. In: Harrison F. W. and Corliss J. O. (eds.): Microscopic anatomy of invertebrates, vo!. 1: Protozoa, pp. 13-159. Wiley-Liss, New York. 23 Vivier E. (1974): Morphology, taxonomy and general biology of the genus Paramecium. In: van Wagtendonk W. J. (ed.) : Paramecium - a current survey, pp. 1- 89. Elsevier, Amsterdam. 24 Vivier E. et Andre J. (1961): Donnees structurales et ultrastructurales nouvelles sur la conjugaison de Paramecium caudatum. J. Protozoo!., 8, 416-426. 25 Watanabe T and Takei K. (1982): Exocytosis defective mutants in Paramecium caudatum. Zoo!' Magazine, 91,580. 26 Wessenberg H. and Antipa G. (1968): Studies on Didinium nasutum. I. Structure and ultrastructure. Protistologica, 4, 427-447. 27 Wessenberg H. and Antipa G. (1970): Capture and ingestion of Paramecium by Didinium nasutum. J. Protozoo!., 17,250-270. 28 Wichterman R. (1953): The biology of Paramecium. Blakiston, New York. 29 Wichterman R. (1986): The biology of Paramecium. 2nd cd. Plenum Press, New York. 30 Wohlfarth-Bottermann K. E. (1953): Experimentelle und elektronenoptische Untersuchungen zur Funktion der Trichocysten von Paramecium caudatum. Arch. Protistenk., 98, 169-226.
Key words: Trichocysts - Predator-prey interaction - Paramecium - Monodinium - Didinium Akio Miyake, Dipartimento di Biologia MCA, Universita di Camerino, Via F. Camerini 2, 62032 Camerino (MC), Italy