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Defence function of pigment granules in Stentor coeruleus
Europ. J. Protistol. 37, 77–88 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ejp
Defence function of pigment granules in Stentor coeruleus Akio Miyake1, *, Terue Harumoto2 and Hideo Iio3 1
Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (MC), Italy; Tel.: ++39 0737 632216 or 403221, Fax.: ++39 0737 632216 or 636216, E-mail: [email protected] 2 Department of Biological Science, Nara Women’s University, 630-8506 Nara, Japan 3 Department of Material Science, Osaka City University, 558-8585 Osaka, Japan Received: 20 October 2000; 7 December 2000. Accepted: 14 December 2000
Pigment granules in Stentor coeruleus are extrusive organelles containing the pigment, stentorin, which provides the blue-green colouration to this ciliate. We studied the defence function of these organelles by 1) observing the interaction between S. coeruleus and the predatory ciliate Dileptus margaritifer; 2) comparing normally-pigmented cells and artificially-bleached cells of S. coeruleus as prey for the predator; and 3) measuring the toxicity of chemically-synthesized stentorin to D. margaritifer, S. coeruleus, and 7 other ciliates. When a Dileptus attacked a Stentor, the Stentor released a mass of bluish material and the Dileptus retreated. Bleached cells of S. coeruleus were more vulnerable than normally pigmented cells to the predator. Stentorin was highly toxic to D. margaritifer (LD50, 0.6–1.0 µg/ml) in the dark, but much less toxic to S. coeruleus (LD50, 90 µg/ml). Under certain conditions, Dileptus was killed by normally pigmented Stentor, but not by bleached ones. We conclude that pigment granules of S. coeruleus function as organelles of defence against D. margaritifer and that the chemical basis of this defence is the pigment stentorin. Key words: Stentor ; Dileptus; Pigment granules; Extrusome; Chemical defence.
Introduction Stentor coeruleus is a blue-green, heterotrich ciliate. The colouration is due to the pigment stentorin contained in pigment granules which are spherical membrane-bounded organelles of 0.3–1.0 µm in diameter mostly localized in the cortex just below the cell surface (Inaba 1959; Newman 1974; Kim et al. 1990, see Tartar 1961; Song 1981 for review). Tartar (1961), who had studied Stentor extensively, suggested two possible functions of stentorin, photoreception and protection against predators. Later works showed that stentorin participates in the photophobic response of this ciliate (Wood *corresponding author
1976; Song et al. 1980, see Song 1981 for review) and since then stentorin has been investigated as a photoreceptor pigment. The possible protective function of stentorin has not been studied, but here we report that this pigment participates in the defence of S. coeruleus against the predatory ciliate Dileptus margaritifer. Stentorin was identified as 2,2′,4,4′,5,5′, 7,7′octahydroxy-3,3′-diisopropylnaphthodianthrone (Tao et al. 1993; Cameron and Riches 1995) and chemically synthesized (Cameron and Riches 1995; Iio et al. 1995). It is a polycyclic aromatic compound related to hypericin, which is the photodynamic toxin of Hypericum, St. John’s Wort (Giese 1980). Stentorin in pigment granules is asso0932-4739/01/37/01-077 $ 15.00/0
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ciated with proteins forming chromoproteins (Kim et al. 1990) and these chromoproteins are also called stentorin. In this work, stentorin indicates the polycyclic aromatic compound, the chromophore of those chromoproteins. S. coeruleus sheds the pigment when responding to various chemical stimuli (Tartar 1961). Pigment granules of S. coeruleus are, therefore, extrusive organelles and are considered to be extrusomes of the mucocyst type (Hausmann 1978; Dragesco 1984). We have shown that pigment granules in Blepharisma japonicum, another coloured heterotrich ciliate, are defence organelles against the predatory ciliate D. margaritifer (Miyake et al. 1990; Harumoto et al. 1998) and suggested that pigment granules in S. coeruleus may function similarly. In this work, we tested this hypothesis by 1) observing the interaction between S. coeruleus and D. margaritifer, 2) comparing normally-pigmented cells and artificially-bleached cells of S. coeruleus as prey for the predator, and 3) measuring the toxicity of chemically-synthesized stentorin against D. margaritifer, S. coeruleus and 7 other ciliates. The results verified the hypothesis. Part of this work has been published in abstract form (Harumoto and Miyake 1994; Harumoto et al. 1995; Miyake and Harumoto, Proc. 4th Asian Conf. Ciliate Biol., Tokyo 1995, pp. 69–71).
Material and methods Ciliates Ciliates and culture methods used in this work are listed in Table 1. Stocks of Stentor coeruleus were collected in Münster, Germany (Mün 1, Mün 13, and Mün 20) and Policoro, Italy (Pol 1). Mün 1 and Mün 13 were larger in size and less deeply coloured than Mün 20 and Pol 1 (the diameter of shrunken cells in the stationary phase: about 300 µm for Mün 1 and Mün 13, 200 µm for Mün 20 and Pol 1). Unless specified, Mün 1 was used. As predator, 3 stocks of the haptorid Dileptus margaritifer, formerly D. anser (Wirnsberger et al. 1984), were used. Stock SHL1 is a hybrid between stocks L and SH both provided by Dr. K. Golinska, Nencki Inst. Exp. Biol., Warsaw. Stock D3-I was provided by Dr. M. Tavrovskaya, Inst. Cytol. Russ. Acad. Sci., St. Petersburg. D3-I was distinctly larger than SHL1 (cells in the stationary phase were about 800 and 500 µm in length and 60 and 50 µm in width, respectively), while stock F1-4, a hybrid between SHL1 and D3-I, was intermediate in size. Intrastock conjugation was not observed in any of them. Unless specified, SHL1 was used.
Table 1. Ciliates and culture methods used in this work. Species (stocks)
Culture methods1
Blepharisma japonicum (R1072)2 Climacostomum virens (W-24) Colpidium sp. (War 1)5 Didinium nasutum (7771)3 Dileptus margaritifer (SHL1, D3-I, F1-4) Euplotes octocarinatus (3-58) Lembadion bullinum (3A) Paramecium tetraurelia (51) Sathrophilus4 sp. (Pisa 7)5 Stentor coeruleus (Mün 1, 13, 20, Pol 1) Stentor polymorphus (Mün 32) Tetrahymena thermophila (210)5
L C (Sathrophilus sp.) L C (P. tetraurelia) C (Sathrophilus sp.) C (T. thermophila) C (Colpidium sp.) L L C (Sathrophilus sp.) C (Sathrophilus sp.) L
1
L, grown on the lettuce medium inoculated with Enterobacter aerogenes (Miyake and Beyer 1973); C, grown on the ciliate in parentheses suspended in SMB. 2 Bangalore strain (Hirshfield et al. 1973). 3 Derived from the ATCC strain 30399 purchased from American Type Culture Collection. 4 Formerly Saprophilus (Corliss 1960). 5 Used only to feed other ciliates.
The ciliates grown on Enterobacter aerogenes in the lettuce medium (L in Table 1) were concentrated by centrifugation, washed by and suspended in SMB-III (1.5 mM NaCl, 0.05 mM KCl, 0.4 mM CaCl2, 0.05 mM MgCl2, 0.05 mM MgSO4, 2 mM Na-phosphate buffer pH 6.8, 2 × 10-3 mM EDTA) (Miyake 1981) (called SMB below), and used after one day. Debris in the culture was removed as described (Miyake and Honda 1976) using a nylon net with a mesh size appropriate to the respective ciliate. Those grown on small ciliates (C in Table 1) were used one day after they had consumed the food. Culture, handling of ciliates and experiments were performed at 24 ± 1°C unless specified.
Bleaching of Stentor The colouration of S. coeruleus was observed with unaided eyes on a white background. This simple method allowed to distinguish between extensively bleached white cells and less-bleached pale-blue cells, both of which looked similar (grayish-brown) in a stereomicroscope with transillumination. Bleaching by high-temperature: Cells grown at temperatures higher than 29 °C gradually lost the bluish colour. At 30°C the bleaching was nearly complete after
Chemical defence in Stentor
10 days, but cells grew more slowly, stopped growing several days later and started dying after several more days. At 29 °C cells turned pale blue after about 10 days and remained so for at least 2 months. Cells continued growing and they looked as healthy as unbleached cells (see also Fig. 1C). Bleaching by caffeine: Following Tartar’s (1972) method, we bleached S. coeruleus by growing cells in the presence of 0.2 mM caffeine (Caffeine anhydrous, Fluka). Cells in the caffeine-containing culture medium multiplied normally except during the first 3–4 days, turned pale blue after several days and remained so until the culture was discontinued after 2 months. Caffeinebleached cells looked as healthy as unbleached cells; they conjugated with unbleached cells of the complementary mating type. Before use, cells were washed 2–3 times by repeating mild centrifugation in SMB and incubated in SMB for 2 hours. Bleaching by lysozyme: Lysozyme, which induces a massive discharge of trichocysts in live cells of Paramecium (Harumoto and Miyake 1991), induced a massive discharge of pigment granules in S. coeruleus. Cells were treated with 50 and 100 µg/ml lysozyme (crystallized lysozyme from hen egg, Boehringer Mannheim) in SMB for 10 minutes, washed with and suspended in SMB as caffeine-treated cells described above. Cells so treated looked pale blue, but otherwise normal (see also Fig. 3C).
Stentorin Chemically-synthesized stentorin (Iio et al. 1995) was dissolved in 75% ethanol (2 mg/ml) and stored in the dark at 2–6 °C. The solution was diluted with SMB at the time of the experiment.
Toxicity-test of stentorin Ten cells of a ciliate were placed in 250 µl of stentorin solution of various concentrations and the number of surviving cells were counted after 24 hours. The LD50 concentration of stentorin for a ciliate was obtained based on the concentration-survival curve of the ciliate (Fig. 4). Unless specified, cells were kept in a dark moist chamber until the time of observation.
Results 1. Observations on the interaction between a Stentor and a Dileptus Dileptus attacks prey with a long flexible toxicysts-bearing proboscis. A single hit with the proboscis can induce an instant total disintegration of small prey like Colpidium (Dragesco 1962) and inflicts a local lysis in large prey such as S. coeruleus
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(Visscher 1923) and Blepharisma japonicum (Miyake et al. 1990). Five cells of Dileptus and 30 cells of Stentor were placed in 250 µl SMB in a depression slide and the outcome of the encounter between a Dileptus and a Stentor was observed in a stereomicroscope. A characteristic interaction occurred when the proboscis of a Dileptus touched a Stentor. If the Stentor was extended, it immediately contracted and, within a second, a mass of bluish material appeared between the Stentor and the proboscis. The Dileptus swam backward often with the bluish material, which was clinging to the proboscis. Sometimes a bluish cloud-like mass was seen rising from the Stentor at or near the site touched by the proboscis. If another Dileptus happened to touch the “blue cloud”, the Dileptus pulled back indicating that the cloud contains a factor which induces backward swimming in Dileptus. Since the Stentor, but not the Dileptus, was blue coloured, it was deduced that the blue cloud contained material derived from the Stentor. The retreated Dileptus often had a less pointed and slightly shorter proboscis and the cell became sluggish. If a Dileptus stayed with a Stentor for several seconds before retreating, these changes were more prominent. In spite of these pathological reactions, a retreated Dileptus sometimes ingested a part or the whole of the bluish material, which the Dileptus carried with its proboscis. In some cases, an attacking Dileptus was surrounded by the blue cloud, sank to the bottom of the container, remained in the cloud and gradually became spherical in the following hour. In most cases, a retreated Dileptus attacked a Stentor again, but as they repeated attacks, the shortening of the proboscis continued. The Dileptus often lost the entire proboscis, became spherical and died in several hours. When other stocks of S. coeruleus and D. margaritifer were used, the result was essentially the same. In D3–I and F1–4 of Dileptus, which are larger than SHL1, the backward swimming and the pathological reactions were slightly less prominent. In Mün 20 and Pol 1 of Stentor, which are smaller but more intensely coloured, the mass of bluish material was more deeply coloured, clung to the proboscis of Dileptus more often and apparently injured Dileptus more severely. These observations indicate that S. coeruleus defends itself against the attack by D. margaritifer and suggest that the blue pigment of S. coeruleus, stentorin, participates in the defence.
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Table 2. Offence-defence interaction between one Stentor (S) and 1, 2, 5, 7 and 10 cells of Dileptus (D) in 100 µl SMB. Mean numbers of surviving cells from 3 experiments. Time
Cells
(day)
1S–1D
1S–2D
1S–5D
1S–7D
1S–10D
S
D
S
D
S
D
S
D
S
D
0
1.0
1.0
1.0
2.0
1.0
5.0
1.0
7.0
1.0
10.0
1
1.0
0.0
0.7
1.0
0.3
3.7
0.0
9.3
0.0
11.3
2
1.0
0.0
0.7
1.0
0.3
4.3
0.0
9.3
0.0
11.6
2. Interaction between a Stentor and various numbers of Dileptus One cell of Stentor was placed with 1, 2, 5, 7 and 10 cells of Dileptus in 100 µl SMB in a depression slide. These cell mixtures, which were called 1S–1D, 1S–2D, 1S–5D, 1S–7D and 1S–10D, respectively, were made in triplicate and observed for 2 days (Table 2). In 1S–7D and 1S–10D, Stentor disappeared and Dileptus slightly increased in number suggesting that Dileptus consumed Stentor as food. On the contrary, in 1S–1D, it was Dileptus that was killed. In 1S–2D and 1S–5D, both Stentor and Dileptus decreased in number probably killing each other. The result indicates that Dileptus can prey on Stentor only when it is numerically superior over Stentor under these conditions, supporting the assumption that Stentor can defend itself against Dileptus. The result also confirms the observation that Stentor can kill Dileptus.
3. Observations on the interaction between a bleached Stentor and a Dileptus Five cells of Dileptus and 30 cells of bleached Stentor were placed in 200 µl SMB in a depression slide and the outcome of the encounter between a Dileptus and a Stentor was observed in a stereomicroscope. When a Dileptus attacked the Stentor, which had been almost completely bleached after growing at 30 °C for 12 days, the Dileptus seldom retreated, immediately started ingesting the Stentor, and continued the predation, while not showing any sign of affliction. A bleached Stentor is thus more vulnerable to the predator than an unbleached one. However, in the Stentor culture kept at 30 °C for
12 days cells were not entirely normal (see Material and Methods), making it difficult to ascribe the observed higher vulnerability to the reduced colouration. If Stentor was only partially bleached by high temperature, caffeine or by lysozyme, cells appeared as healthy as unbleached ones (see Material and Methods). The interaction between such a partially bleached Stentor and a Dileptus was qualitatively the same as the one between an unbleached Stentor and a Dileptus; the Dileptus retreated after attacking the Stentor and looked afflicted. However, both the behavioural and pathological reactions of the Dileptus looked weaker, suggesting that the partially bleached Stentor is more vulnerable to the predator than the unbleached one. This assumption was tested in the following experiments.
4. Interaction between Dileptus and partially bleached Stentor Stentor was partially bleached by 3 different methods, high temperature, caffeine and lysozyme treatment, and compared with unbleached Stentor as prey for Dileptus. High-temperature bleached Stentor Stentor was bleached by growing at 29 °C for 15 days. Cells were pale blue but looked as healthy as unbleached cells. Five cells of bleached and unbleached Stentor were placed with 5 cells of Dileptus in 250 µl SMB in a depression slide in 21 duplicates and observed for 48 hours (Fig. 1A–B). Both the bleached and unbleached Stentor decreased in number, but the decrease was more
Chemical defence in Stentor
extensive in the bleached one (Fig. 1A). After 48 hours, survival rates were 1% and 85% for the bleached and unbleached Stentor, respectively. The bleached Stentor is, therefore, more vulnerable to predation by Dileptus. The Dileptus mixed with unbleached Stentor decreased in number indicating that it met with a strong counter-attack from the Stentor (Fig. 1B). On the contrary, the Dileptus mixed with the bleached Stentor multiplied, indicating that the Dileptus consumed the Stentor as food (Fig. 1B). In parallel with these experiments, the bleached and unbleached cells of Stentor were fed (Fig. 1C). The bleached cells were not slower in growth than the unbleached cells, both dividing about twice in 48 hours, and supporting the observation that the bleached cells were as healthy as unbleached cells. Caffeine-bleached Stentor Stentor was bleached by growing in the presence of caffeine (0.2 mM) for 7 days. Bleached cells were apparently normal except in colouration (Material and Methods). Five cells of bleached and unbleached Stentor, were placed in 250 µl SMB with 10 cells of Dileptus in a depression slide in 21 duplicates and observed for 48 hours (Fig. 2A–B). Bleached Stentor steadily decreased in number while unbleached Stentor changed little in number (Fig. 2A) indicating that the bleached Stentor is more vulnerable to predation by Dileptus.
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The Dileptus with unbleached Stentor decreased in number indicating that the Dileptus met with a strong counter-attack from Stentor. The Dileptus with bleached Stentor decreased a little in number at the beginning but then multiplied, indicating that the Dileptus preyed on the bleached Stentor after an initial suffering from the counter-attack by the Stentor. Lysozyme-bleached Stentor In the first experiment, Stentor was bleached by suspending cells in 50 and 100 µg/ml lysozyme for 10 minutes. In both treatments, bleached cells looked similarly pale blue. Unbleached cells for the control were treated in the same way including the washing by repeated centrifugation (Material and Methods) except that they were suspended for 10 minutes in SMB instead of the lysozyme solution. A Stentor, either bleached or unbleached, was placed with a Dileptus in 50 µl SMB in a depression slide in 20 duplicates and observed for 3 days (Table 3). After 2 days, unbleached Stentor survived in 7 mixtures, while bleached Stentor survived only in 1 mixture. Bleached Stentor was killed in all 20 mixtures in 3 days not killing Dileptus at all, while unbleached Stentor survived in 4 mixtures killing Dileptus in all of them. The bleached Stentor is, therefore, more vulnerable to Dileptus than an unbleached one.
Fig. 1. Effect of partial bleaching of Stentor coeruleus by high temperature (29 °C, 15 days) on the offence-defence interaction between S. coeruleus and Dileptus margaritifer. The number of Stentor and that of Dileptus are plotted in A and B, respectively, against the time after mixing 5 cells of Dileptus with 5 bleached (s–––s)* and 5 unbleached (d–––d)** cells of Stentor. In C, bleached (s–––s)* and unbleached (d–––d)** cells of Stentor were compared for their ability to multiply by feeding 5 cells. Means of 21 experiments with SE. *Solid line, unfilled circles; **Solid line, filled circles.
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Table 3. Effect of partial bleaching of Stentor coeruleus by lysozyme on the offence-defence interaction between S. coeruleus and Dileptus margaritifer in 20 oneto-one mixtures. Stentor
Survived cells Time (day) 0
1
2
3
Unbleached
Stentor Dileptus
20 20
15 20
7 20
4 16
Fig. 2. Effect of partial bleaching of Stentor coeruleus by caffeine (0.2 mM, 7 days) on the offence-defence interaction between S. coeruleus and Dileptus margaritifer. The number of Stentor and that of Dileptus are plotted in A and B, respectively, against the time after mixing 10 cells of Dileptus with 5 bleached (s–––s)* and 5 unbleached (d–––d)** cells of Stentor. Means of 21 experiments with SE. *Solid line, unfilled circles; ** Solid line, filled circles.
Bleached by 50 µg/ml lysozyme
Stentor Dileptus
20 20
7 20
0 20
0 20
Bleached by 100 µg/ml lysozyme
Stentor Dileptus
20 20
10 20
1 20
0 20
Significantly, the Stentor treated with 100 µg/ml lysozyme was not more vulnerable than the one treated with 50 µg/ml, in spite of the fact that the chemically-induced abnormality is usually more extensive at higher concentrations of the inducing agent. Since both treatments were the same in their effect on the bleaching (see above), the result supports the assumption that the higher vulnerability of the bleached Stentor is mainly due to the bleaching.
In the second experiment, Stentor was bleached by suspending cells in 50 µg/ml lysozyme for 10 min. Unbleached Stentor was treated in the same way except that they were suspended for 10 minutes in SMB instead of the lysozyme solution. Lysozyme-treated Stentor was distinctly paler than untreated cells, but looked as healthy as untreated cells. Five cells of either bleached or unbleached Stentor were placed with 5 cells of Dileptus in 250 µl SMB in a depression slide in 21 duplicates and
Fig. 3. Effect of partial bleaching of Stentor coeruleus by lysozyme (50 µg/ml, 10 mins) on the offence-defence interaction between S. coeruleus and Dileptus margaritifer. The number of Stentor and that of Dileptus are plotted in A and B, respectively, against the time after mixing 5 cells of Dileptus with 5 bleached (s–––s)* and 5 unbleached (d–––d)** Stentor cells. In C, bleached (s–––s)* and unbleached (d–––d)** cells of Stentor were compared for their ability to multiply by feeding 5 cells. Means of 21 experiments with SE. *Solid line, unfilled circles; **Solid line, filled circles.
Chemical defence in Stentor
83
Fig. 4. Lethal effect of stentorin on various ciliates. A: Lembadion bullinum. B: Paramecium tetraurelia. C: Dileptus margaritifer stock SHL1. D: Dileptus margaritifer stock D3-I. E: Didinium nasutum. F: Euplotes octocarinatus. G: Stentor polymorphus. H: Stentor coeruleus. I: Blepharisma japonicum. J: Climacostomum virens. Mean numbers of surviving cells of 3 experiments with SE.
observed for 28 hours (Fig. 3A–B). In the mixture of bleached Stentor and Dileptus, Stentor decreased in number (Fig. 3A) while Dileptus increased in number (Fig. 3B), indicating that Dileptus preyed on Stentor. On the contrary, in the mixture of Dileptus and unbleached Stentor, the number of Stentor did not decrease (Fig. 3A) while the number of Dileptus decreased (Fig. 3B), indicating that the Stentor not only defended itself from attack by Dileptus, but also effectively counter-attacked the predator. In parallel with these experiments, bleached and unbleached cells of Stentor were compared in their ability to multiply (Fig. 3C). They were indistin-
guishable, both dividing once in 28 hours, supporting the observation that bleached cells were as healthy as unbleached cells.
5. Toxicity of stentorin to Dileptus margaritifer, Stentor coeruleus and other ciliates Behavioural and pathological responses of Dileptus to stentorin Ten cells of Dileptus were placed in 500 µl SMB containing various concentrations of stentorin in a depression slide and observed in a stereomicroscope. At 5–20 µg/ml of stentorin, all cells immedi-
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A. Miyake, T. Harumoto and H. Iio
ately started swimming backward. At 5 µg/ml, cells became sluggish after about 1 minute of backward swimming and their proboscis started degenerating 1–2 minutes later. At 20 µg/ml, the backward swimming lasted only several seconds. Cells then became sluggish and their proboscis started degenerating within a minute. No striking response was observed at concentrations lower than 1 µg/ml. In stock D3-I, the response of cells to stentorin was similar, but they stopped swimming for a few seconds before starting the backward swimming. These responses of Dileptus to stentorin, the backward swimming, sluggishness and degeneration of the proboscis, are quite similar to the reactions of Dileptus after attacking Stentor (Results 1), suggesting that the behavioural and pathological reactions of Dileptus in the Dileptus-Stentor interaction are evoked by stentorin released by Stentor. More quantitative study on the toxicity of stentorin was carried out on the lethal effect. Lethal effect of stentorin on Dileptus and other ciliates The toxicity of stentorin to D. margaritifer (stocks SHL1 and D3-I) and 8 other ciliates was tested as described in Material and Methods (Fig. 4). The LD50 concentration of stentorin for these ciliates, based on the data in Fig. 4, are listed in Table 4. These values range from 0.1 µg/ml (Lembadion) to >100 µg/ml (Blepharisma, Climacostomum). The LD50 for Stentor coeruleus (90 µg/ml) is about
Table 4. Lethal dose 50% (LD50 ) of stentorin for ciliates. Species and stocks
LD50 of stentorin (µg/ml)
Lembadion bullinum Paramecium tetraurelia Dileptus margaritifer SHL1 Dileptus margaritifer D3-I Didinium nasutum Euplotes octocarinatus Stentor polymorphus Stentor coeruleus Blepharisma japonicum Climacostomum virens1
0.1 0.5 0.6 0.9 1.1 2.3 5.0 90.0 >100.0 >100.0
1 “White” cells lacking the colouration due to symbiotic Chlorella.
100 times higher than the LD50 for Dileptus (0.6 and 0.9 µg/ml). The four heterotrichs tested are all more resistant than other ciliates, a possible explanation of which is given in the Discussion. In Dileptus, stock D3-I is more resistant than stock SHL1 (Fig. 4C–D, Table 4), indicating that there is also intraspecific variation in the sensitivity to stentorin. Effect of light on the toxicity of stentorin Since stentorin is a photodynamic pigment (Yang et al. 1986), we examined the effect of light on the toxicity of stentorin. The LD50 of stentorin for Dileptus (D3-I) was measured in the light and in the dark at the same time. The test in the dark was carried out as described in Material and Methods. The test in the light was carried out in the same way except that Dileptus was kept in a glass moist chamber placed under a 40W cylindrical fluorescence bulb at a distance of 40 cm. The LD50 of stentorin in the dark was about 5 times higher than that in the light (Fig. 5), indicating that photodynamic action participates in the toxicity of stentorin in the light.
Conclusion Fig. 5. Effect of light on the lethal effect of stentorin on Dileptus margaritifer (stock D3-I). s–––s*, L: Experiment in the light. d–––d**, D: Experiment in the dark. Mean numbers of surviving cells of 3 experiments with SE. *Solid line, unfilled circles; **Solid line, filled circles.
Observations on the interaction between Dileptus margaritifer and Stentor coeruleus (Results 1 and 2) show that Dileptus attacks Stentor and that Stentor defends itself against the predator. The observation also suggests that the defence of Stentor
Chemical defence in Stentor
is based on the blue pigment, stentorin. This hypothesis is supported by observations on the interaction between bleached Stentor and Dileptus (Results 3), and more strongly by the experimental result that bleaching of Stentor by three different methods made this ciliate more vulnerable to Dileptus without inducing any detectable abnormalities except in colouration (Results 4). The hypothesis is also supported by the result that chemically-synthesized stentorin induced all the behavioural and pathological responses of Dileptus observed in the Dileptus-Stentor interaction, except the ingestion of and the entanglement with the material released from Stentor (Result 5). In addition, the result that the pigment was about 100 times more toxic to Dileptus than to Stentor is consistent with the hypothesis (Results 5). We, therefore, conclude that stentorin participates in the defence of Stentor coeruleus against the predatory ciliate Dileptus margaritifer. Since stentorin is localized in pigment granules of S. coeruleus, we also conclude that pigment granules of S. coeruleus have the function of defence.
Discussion Function of pigment granules in Stentor coeruleus and other heterotrich ciliates With the above conclusion, this work verifies the old assumption of Tartar (1961) that the pigment in Stentor coeruleus might have some protective value against predators. His assumption was based on 1) the unpublished work of K. M. Møller and A. H. Whiteley (published as appendix in (Møller 1962)) that the alcohol-extracted pigment of some races of Stentor coeruleus was photolethal to Paramecium caudatum and Colpidium, and 2) the speculation that the red pigment of Stentor igneus might be identical to the red pigment of Blepharisma known to be toxic to certain other protozoa (Giese 1949). Møller (1962) considered the possible defence function of pigment granules as a somewhat far-fetched explanation for their function. Giese (1973a) once assumed that the red pigment of Blepharisma might have a defence function against predators, but, having no direct evidence for this function, he thought that the most likely function of the pigment is protection against far UV radiation (Giese 1973a, 1973b, 1981).
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Later, Estève (1982) compared dozens of ciliates as prey for Dileptus visscheri, Dileptus sp. and Lacrymaria olor and noted that these predators showed “reticence” to ingest pigmented heterotrichs, including S. coeruleus. He interpreted the phenomenon as food selection in predators, but the result could be interpreted also as an indication of the protective function of pigments in these heterotrichs. More recently, the hypothesis on the defence function of pigment granules in B. japonicum was experimentally verified (Miyake et al. 1990; Harumoto et al. 1998), and this prompted us to study the defence function of the pigment in S. coeruleus. Tartar (1961) also thought that stentorin might have a photoreceptor function. This assumption was verified more quickly. It has been shown that stentorin is the photoreceptor pigment for the step-up photophobic response and negative phototaxis of S. coeruleus (Song et al. 1980, see Song 1981; Song et al. 1991 for review). Later, blepharismin was also shown to function as a photoreceptor for the photophobic behaviour of B. japonicum (Scevoli et al. 1987; Matsuoka et al. 1992, 1995; Checcucci et al. 1993; see Ghetti 1991 for review). Thus pigment granules in B. japonicum and S. coeruleus have two functions, defence against predators and photoreception, suggesting that pigment granules in other heterotrichs also have these functions. Harumoto et al. (1998) discussed the two functions in B. japonicum and proposed that the primary function of pigment granules is chemical defence. The same argument can be applied to S. coeruleus. Indeed, some features of pigment granules of S. coeruleus, such as the presence in large numbers throughout the cell surface and the discharge of their content in responding to various external stimuli, conform better to the defence function. Tartar (1961) tested 8 races of S. coeruleus for the negative response to daylight and found that two showed a strong response, two showed no response and the rest showed intermediate response. The result suggests that the photophobic response of S. coeruleus is the subject of a wide variation among stocks. On the other hand, in the present work all of the 4 stocks of S. coeruleus tested strongly repelled the predator. These results are consistent with the assumption that chemical defence is the primary function of pigment granules.
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Relevant to this question is the presence of many colourless or apparently colourless heterotrichs including Stentor polymorphus (Tartar 1961) and Blepharisma hyalinum (Larsen and Nilsson 1983). They have colourless or colourlesslooking cortical vesicles, which are morphologically similar to pigment granules. This raises questions; whether their cortical vesicles contain a minute amount of pigment for photoreception, and whether they contain a colourless toxin for defence. The latter question was examined in recent studies on the heterotrich Climacostomum virens, which has colourless cortical vesicles (Peck et al. 1975). Masaki et al. (1999) isolated a toxic substance from the extract of C. virens, identified it as 1, 3-dihydroxy-5-[(Z)-2′-nonenyl]benzene, named it climacostol and suggested that climacostol serves for defence against predators. It was subsequently demonstrated that cortical vesicles of C. virens function as organelles for chemical defence against the predatory ciliate Dileptus margaritifer and that the chemical basis of this defence is climacostol (Miyake A. Buonanno F. and Saltalamacchia P.; Abstracts, 3rd Eur. Congr. Protistol., 9th Eur. Conf. Ciliate Biol., Helsingør, Denmark, 1999 p.50; personal communication). Assuming that colourless cortical vesicles and pigment granules in heterotrichs are homologous organelles, and hence that pigment granules are pigmented cortical vesicles, this work on C. virens suggests that chemical defence is a general function of cortical vesicles in heterotrichs. Masaki et al. (1999) suggest that the climacostol is biochemically related to stentorin. The possibility that defence toxins in heterotrichs are chemically related to each other, provides a hint to understand the result that the four heterotrichs tested are all more resistant than other ciliates to stentorin (Fig. 4, Table 4).
Defence against predators in Stentor coeruleus S. coeruleus is a large contractile sedentary ciliate. It usually attaches to a substratum with the posterior end and extends itself. During swimming, it is often contracted to some extent. These cells rapidly contract when responding to various stimuli, e.g., an attack by Dileptus margaritifer. Both the large size and the contractility have protective values against the predator. Be-
cause of its size, S. coeruleus can survive the first hit with the proboscis of D. margaritifer, which instantly kills small ciliates like Tetrahymena pyriformis. The large size also makes it possible to leave a piece of destroyed cytoplasm as a bite for the predator to help in escaping. The contraction may serve for separating Stentor from the predator and also from the toxin released by the predator. It may also provide a mechanical stimulus to the predator to induce a retreating reaction. In addition, S. coeruleus can defend itself chemically by means of toxin-containing extrusomes, pigment granules, as shown in this work. Reactions of Dileptus in the Dileptus-Stentor interaction described in this work are backward swimming, slowing down, ingestion of and entanglement with the material released from Stentor, degeneration of the proboscis, pathological rounding of the cell, and death. They are all helpful for Stentor in escaping from the predator. The lethal reaction of Dileptus is probably a laboratory artifact, however. It took several hours under the condition in which Dileptus is confined in a small space with Stentor and, therefore, may not occur in nature where more space is available for dispersion of cells and dilution of toxins. On the other hand, the partial degeneration of the proboscis occurs more quickly and is important in the defence of Stentor against Dileptus in nature. Most important for the defence appears to be the behavioural reaction of Dileptus. The backward swimming of attacking Dileptus provides Stentor a good chance to escape. The slowing down of the retreated predator further facilitates the escape. If the retreated predator ingests a piece of the material released from Stentor or if it is entangled with it, Stentor gains even longer time to escape. Since the retreating and pathological reactions of Dileptus are all induced by chemically synthesized stentorin, we assume that stentorin in pigment granules is responsible for these reactions. The possibility that other unknown toxins also participate in these reactions is not entirely excluded, however. The backward swimming of Dileptus may be induced by the sudden contraction of Stentor, but a fully contracted Stentor also induced the backward swimming indicating that stentorin can induce this reaction without any help of the contraction. On the other hand, the ingestion of and entanglement with the material released from Stentor requires the physical integrity that stentorin alone cannot provide.
Chemical defence in Stentor
Pigment granules in S. coeruleus contain glycoproteins and a lipoprotein (Song 1981). Stentorin in pigment granules associates with glycoproteins to form chromoproteins (Kim et al. 1990), which are thought to function as photoreceptors (Kim et al. 1990; Song et al. 1990). These proteins may serve, together with other components in pigment granules, for maintaining the physical integrity of the discharged material of pigment granules. If so, these proteins play their own role in the defence function of pigment granules. Stentorin has two kinds of toxicity, the phototoxicity, which is light dependent, and the intrinsic toxicity, which is independent of light. Since stentorin is, like blepharismin (Giese 1973a), a photodynamic pigment (Yang et al. 1986), the toxicity of stentorin in the light is partly due to photodynamic action, i.e., photo-oxidation mediated by a photosensitizer pigment. Indeed, stentorin was about 5 times more toxic to D. margaritifer even under moderately illuminated conditions (Results 5, Fig. 5). Since the behavioural and pathological reactions of Dileptus reported in this work were observed under ordinary illumination, both phototoxicity and intrinsic toxicity participated in these reactions. When the Dileptus-Blepharisma interaction was observed under red light, which is scarcely absorbed by blepharismin, the reactions of Dileptus were virtually the same as those observed under ordinary illumination, indicating that the intrinsic toxicity of blepharismin alone can induce these reactions (Harumoto et al. 1998). Since stentorin is chemically closely related to blepharismin and since the pigment is toxic in the dark (Results 5), it is inferred that the intrinsic toxicity of stentorin can induce the behavioural reactions of Dileptus. The intrinsic toxicity of stentorin makes it possible to maintain the defence function at night. Even in daytime, it is probably important for the defence, as S. coeruleus tends to stay in shady places because of the photophobic behaviour. Little is known about the molecular mechanism of the intrinsic toxicity of stentorin. The problem of how pigment granules are discharged in the Dileptus-Stentor interaction can be inferred, based on the study on the DileptusBlepharisma interaction. Stereomicroscopic observations on the Dileptus-Stentor interaction indicate that the discharge of pigment by Stentor is localized at and near the site touched by the proboscis of Dileptus. This is quite similar to that which occurs in the Blepharisma-Dileptus inter-
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action. In the latter, observations in a scanning electron microscope at the moment of the attack by Dileptus (Harumoto et al. 1998) showed that pigment granules of Blepharisma were discharged at the area adjacent to the site, which was physically damaged by the attack. At this area, the cell surface was morphologically intact except for the presence of many round depressions, each of which was thought to have been produced by the discharge of a pigment granule. The discharge of pigment granules occurs, therefore, at the intact cell surface as a response to the stimulus coming from the attack by Dileptus. The discharge of pigment granules in S. coeruleus attacked by Dileptus probably occurs in the same way. Interestingly, S. coeruleus is apparently defenceless against the predatory ciliate Spathidium sp. (Kuhlmann H.-W. personal communication, our unpublished observation). Studies on the interaction between these two ciliates will provide new insight for understanding the defence mechanism of S. coeruleus against predators. Acknowledgement: We thank Dr. M. Tavrovskaya, Inst. Cytol. Russ. Acad. Sci., St. Petersburg, for the strain D3-I of Dileptus margaritifer, Dr. K. Golinska, Nencki Inst. Exp. Biol., Warsaw, for strains L and SH of D. margaritifer, Drs. K. Heckmann and H.-W. Kuhlmann, Inst. Gen. Zool. & Genet., Univ. Münster, for help in collecting Stentor stocks, and Ms. V. Rivola and Mr. P. Saltalamacchia, Univ. Camerino, for technical assistance. This work was supported by Italian CNR and MURST.
References Cameron D. W. and Riches A. G. (1995): Synthesis of stentorin. Tetrahedron Lett. 36, 2331–2334. Checcucci G., Damato G., Ghetti F. and Lenci F. (1993): Action spectra of the photophobic response of the blue and red forms of Blepharisma japonicum. Photochem. Photobiol. 57, 686–689. Corliss J. O. (1960): The problem of homonyms among generic names of ciliated protozoa, with proposal of several new names. J. Protozool. 7, 269–278. Dragesco J. (1962): Capture et ingestion des proies chez les infusoires ciliés. Bull. Biol. Fr. Belg. 96, 123–167. Dragesco J. (1984): Organites sous-cuticulaires éjectables, éjectisomes (trichocystes). In: Grassé P.-P. (ed.): Traité de zoologie II. Infusoires ciliés I. pp.181–199. Masson, Paris. Estève J.-C. (1982): Défaillance de controle du alimentaire chez les ciliés gymnostomes Dileptus et Lacrymaria. Protistologica 18, 517–526. Giese A. C. (1949): A cytotoxin from Blepharisma. Biol. Bull. 97, 145–149.
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Giese A. C. (1973a): The pigment blepharismin and photosensitivity. In: Giese A. C. (ed.): Blepharisma, pp. 266–303. Stanford Univ. Press, Stanford, California. Giese A. C. (1973b): Conclusion. In: Giese A. C.(ed.): Blepharisma, pp. 333–338. Stanford Univ. Press, Stanford, California. Giese A. C. (1980) Hypericism. Photochem. Photobiol. Rev. 5, pp. 229–255. Giese A. C. (1981) The photobiology of Blepharisma. Photochem. Photobiol. Rev. 6, 139–180. Ghetti F. (1991): Photoreception and photomovements in Blepharisma japonicum. In: Lenci F., Ghetti F., Colombetti G., Häder D.-P. and Song P.-S. (eds.): Biophysics of photoreceptors and photomovements in microorganisms, pp. 257–265. Prenum Press, New York. Harumoto T. and Miyake A. (1991): Defensive function of trichocysts in Paramcium. J. Exp. Zool. 260, 84–92. Harumoto T. and Miyake A. (1994): Defensive function of pigment granules in Stentor. Zool. Sci. 11, Suppl. 24. Harumoto T., Iio H., Zenfuku K. and Miyake A. (1995): Defensive function of pigment granules in Stentor. Zool. Sci. 12, Suppl. 26. Harumoto T., Miyake A., Ishikawa N., Sugibayashi R., Zenfuku K. and Iio H. (1998): Chemical defense by means of pigmented extrusomes in the ciliate Blepharisma japonicum. Europ. J. Protistol. 34, 458–470. Hausmann K. (1978): Extrusive organelles in protists. Int. Rev. Cytol. 52, 197–276. Hirshfield H. I., Isquith I. R. and DiLorenzo A. M. (1973): Classification, distribution, and evolution. In: Giese A. C. (ed.): Blepharisma, pp. 304–332. Stanford Univ. Press, Stanford, California. Iio H. Zenfuku K. and Tokoroyama T. (1995): A facile synthesis of stentorin, the photoreceptor of Stentor coeruleus. Tetrahedron Lett. 36, 5921–5924. Inaba F. (1959): Electron-microscopic study on the fine structure of Stentor coeruleus. Bull. Biol. Soc. Hiroshima Univ. 10, 35–43. (in Japanese with English summary) Kim I.-H., Rhee J.-S., Huh J.-W.,Florell S., Faure B., Lee K.W., Kahsai T., Song P.-S., Tamai N., Yamazaki T. and Yamazaki I. (1990): Structure and function of the photoreceptor stentorins in Stentor coeruleus. I. Partial characterization of the photoreceptor organelle and stentorins. Biochem. Biophys. Acta. 1040, 43–57. Larsen H. F. and Nilsson J. R. (1983): Is Blepharisma hyalinum truly unpigmented? J. Protozool. 30, 90–97. Masaki M. E., Harumoto T., Terazima M. N., Miyake, A., Usuki, Y. and Iio H. (1999): Climacostol, a defense toxin of the heterotrich ciliate Climacostomum virens against predators. Tetrahedron Lett. 40: 8227–8229. Matsuoka T., Matsuoka S., Yamaoka Y., Kuriu T., Watanabe Y., Takayanagi M., Kato Y. and Taneda K. (1992): Action spectra for step-up photophobic response in Blepharisma. J. Protozool. 39, 498–502. Matsuoka T., Watanabe Y., Sagara Y., Takayanagi M. and Kato Y. (1995): Additional evidence for blepharismin photoreceptor pigment mediating step-up photophobic response of unicellular organism, Blepharisma. Photochem. Photobiol. 62, 190–193. Miyake A. (1981): Cell interaction by gamones in Blepharisma. In: O’Day D. H. and Horgen P. A. (eds.): Sexual inter-
action in eukaryotic microbes, pp. 95–129. Academic Press, New York. Miyake A. and Beyer J. (1973): Cell interaction by means of soluble factors (gamones) in conjugation of Blepharisma intermedium. Exp. Cell Res. 76, 15–24. Miyake A. and Honda H. (1976): Cell union and protein synthesis in conjugation of Blepharisma. Exp. Cell Res. 100, 31–40. Miyake A., Harumoto T., Salvi B. and Rivola V. (1990): Defensive function of pigment granules in Blepharisma. Europ. J. Protistol. 25, 310–315. Møller K. M. (1962): On the nature of stentorin. C. R. Trav. Lab. Carlsberg Ser. Chim. 32, 471–498. Newman E. (1974): Scanning electron microscopy of the cortex of the ciliate Stentor coeruleus. A view from the inside. J. Protozool. 21, 729–737. Peck R., Pelvat B., Bolivar I. and Haller G de (1975): Light and electron microscopic observation on the heterotrich ciliate Climacostomum virens. J. Protozool. 22, 368–385. Scevoli P., Bisi F., Colombetti G., Ghetti F., Lenci F. and Passarelli V. (1987): Photomotile responses of Blepharisma japonicum I: Action spectra determination and timeresolved fluorescence of photoreceptor pigments. J. Photochem. Photobiol., B: Biol. 1, 75–84. Song P.-S. (1981): Photosensory transduction in Stentor coeruleus and related organisms. Biochim. Biophys. Acta 639, 1–29. Song P.-S., Häder D. P. and Poff K. L. (1980): Step-up photophobic response in the ciliate, Stentor coeruleus. Arch. Microbiol. 126, 181–186. Song P.-S., Kim I.-H., Florell S., Tamai N., Yamazaki T. and Yamazaki I. (1990): Structure and function of the photoreceptor stentorins in Stentor coeruleus. II. Primary photoprocess and picosecond time-resolved fluorescence. Biochim. Biophys. Acta 1040, 58–65. Song P.-S., Kim I.-H., Rhee J. S., Huh J. W., Florell S., Faure B., Lee K. W., Kahsai T., Tamai N., Yamazaki T. and Yamazaki I. (1991): Photoreception and photomovements in Stentor coeruleus. In: Lenci F., Ghetti F., Colombetti G., Häder D.-P. and Song P.-S. (eds.): Biophysics of photoreceptors and photomovements in microorganisms, pp. 267–279. Plenum Press, New York. Tao N., Orlando M., Hyon J.-S., Gross M. and Song P.-S. (1993): A new photoreceptor molecule from Stentor coeruleus. J. Am. Chem. Soc. 115, 2526–2528. Tartar V. (1961): The biology of Stentor. Pergamon Press, New York. Tartar V. (1972): Caffeine bleaching of Stentor coeruleus. J. Exp. Zool. 181, 245–252. Visscher J. P. (1923): Feeding reactions in the ciliate Dileptus gigas with special reference to the function of trichocysts. Biol. Bull. 45, 113–143. Wirnsberger E., Foissner W. and Adam H. (1984): Morphologie und intraciliatur von Perispira pyriformis nov. spec., Cranotheridium foliosus (Foissner, 1983) nov. comb. und Dileptus anser (O. F. Müller, 1786) (Protozoa, Ciliophora). Arch. Protistenkd. 128, 305–317. Wood D. C. (1976): Action spectrum and electrophysiological response correlated with the photophobic response of Stentor coeruleus. Photochem. Photobiol. 24, 261–266. Yang K. C., Prusti R. K., Walker E. B., Song P.-S., Watanabe M. and Furuya M. (1986): Photodynamic action in Stentor coeruleus sensitized by endogenous pigment stentorin. Photochem. Photobiol. 43, 305–310.
Defence function of pigment granules in Stentor coeruleus Akio Miyake1, *, Terue Harumoto2 and Hideo Iio3 1
Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (MC), Italy; Tel.: ++39 0737 632216 or 403221, Fax.: ++39 0737 632216 or 636216, E-mail: [email protected] 2 Department of Biological Science, Nara Women’s University, 630-8506 Nara, Japan 3 Department of Material Science, Osaka City University, 558-8585 Osaka, Japan Received: 20 October 2000; 7 December 2000. Accepted: 14 December 2000
Pigment granules in Stentor coeruleus are extrusive organelles containing the pigment, stentorin, which provides the blue-green colouration to this ciliate. We studied the defence function of these organelles by 1) observing the interaction between S. coeruleus and the predatory ciliate Dileptus margaritifer; 2) comparing normally-pigmented cells and artificially-bleached cells of S. coeruleus as prey for the predator; and 3) measuring the toxicity of chemically-synthesized stentorin to D. margaritifer, S. coeruleus, and 7 other ciliates. When a Dileptus attacked a Stentor, the Stentor released a mass of bluish material and the Dileptus retreated. Bleached cells of S. coeruleus were more vulnerable than normally pigmented cells to the predator. Stentorin was highly toxic to D. margaritifer (LD50, 0.6–1.0 µg/ml) in the dark, but much less toxic to S. coeruleus (LD50, 90 µg/ml). Under certain conditions, Dileptus was killed by normally pigmented Stentor, but not by bleached ones. We conclude that pigment granules of S. coeruleus function as organelles of defence against D. margaritifer and that the chemical basis of this defence is the pigment stentorin. Key words: Stentor ; Dileptus; Pigment granules; Extrusome; Chemical defence.
Introduction Stentor coeruleus is a blue-green, heterotrich ciliate. The colouration is due to the pigment stentorin contained in pigment granules which are spherical membrane-bounded organelles of 0.3–1.0 µm in diameter mostly localized in the cortex just below the cell surface (Inaba 1959; Newman 1974; Kim et al. 1990, see Tartar 1961; Song 1981 for review). Tartar (1961), who had studied Stentor extensively, suggested two possible functions of stentorin, photoreception and protection against predators. Later works showed that stentorin participates in the photophobic response of this ciliate (Wood *corresponding author
1976; Song et al. 1980, see Song 1981 for review) and since then stentorin has been investigated as a photoreceptor pigment. The possible protective function of stentorin has not been studied, but here we report that this pigment participates in the defence of S. coeruleus against the predatory ciliate Dileptus margaritifer. Stentorin was identified as 2,2′,4,4′,5,5′, 7,7′octahydroxy-3,3′-diisopropylnaphthodianthrone (Tao et al. 1993; Cameron and Riches 1995) and chemically synthesized (Cameron and Riches 1995; Iio et al. 1995). It is a polycyclic aromatic compound related to hypericin, which is the photodynamic toxin of Hypericum, St. John’s Wort (Giese 1980). Stentorin in pigment granules is asso0932-4739/01/37/01-077 $ 15.00/0
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ciated with proteins forming chromoproteins (Kim et al. 1990) and these chromoproteins are also called stentorin. In this work, stentorin indicates the polycyclic aromatic compound, the chromophore of those chromoproteins. S. coeruleus sheds the pigment when responding to various chemical stimuli (Tartar 1961). Pigment granules of S. coeruleus are, therefore, extrusive organelles and are considered to be extrusomes of the mucocyst type (Hausmann 1978; Dragesco 1984). We have shown that pigment granules in Blepharisma japonicum, another coloured heterotrich ciliate, are defence organelles against the predatory ciliate D. margaritifer (Miyake et al. 1990; Harumoto et al. 1998) and suggested that pigment granules in S. coeruleus may function similarly. In this work, we tested this hypothesis by 1) observing the interaction between S. coeruleus and D. margaritifer, 2) comparing normally-pigmented cells and artificially-bleached cells of S. coeruleus as prey for the predator, and 3) measuring the toxicity of chemically-synthesized stentorin against D. margaritifer, S. coeruleus and 7 other ciliates. The results verified the hypothesis. Part of this work has been published in abstract form (Harumoto and Miyake 1994; Harumoto et al. 1995; Miyake and Harumoto, Proc. 4th Asian Conf. Ciliate Biol., Tokyo 1995, pp. 69–71).
Material and methods Ciliates Ciliates and culture methods used in this work are listed in Table 1. Stocks of Stentor coeruleus were collected in Münster, Germany (Mün 1, Mün 13, and Mün 20) and Policoro, Italy (Pol 1). Mün 1 and Mün 13 were larger in size and less deeply coloured than Mün 20 and Pol 1 (the diameter of shrunken cells in the stationary phase: about 300 µm for Mün 1 and Mün 13, 200 µm for Mün 20 and Pol 1). Unless specified, Mün 1 was used. As predator, 3 stocks of the haptorid Dileptus margaritifer, formerly D. anser (Wirnsberger et al. 1984), were used. Stock SHL1 is a hybrid between stocks L and SH both provided by Dr. K. Golinska, Nencki Inst. Exp. Biol., Warsaw. Stock D3-I was provided by Dr. M. Tavrovskaya, Inst. Cytol. Russ. Acad. Sci., St. Petersburg. D3-I was distinctly larger than SHL1 (cells in the stationary phase were about 800 and 500 µm in length and 60 and 50 µm in width, respectively), while stock F1-4, a hybrid between SHL1 and D3-I, was intermediate in size. Intrastock conjugation was not observed in any of them. Unless specified, SHL1 was used.
Table 1. Ciliates and culture methods used in this work. Species (stocks)
Culture methods1
Blepharisma japonicum (R1072)2 Climacostomum virens (W-24) Colpidium sp. (War 1)5 Didinium nasutum (7771)3 Dileptus margaritifer (SHL1, D3-I, F1-4) Euplotes octocarinatus (3-58) Lembadion bullinum (3A) Paramecium tetraurelia (51) Sathrophilus4 sp. (Pisa 7)5 Stentor coeruleus (Mün 1, 13, 20, Pol 1) Stentor polymorphus (Mün 32) Tetrahymena thermophila (210)5
L C (Sathrophilus sp.) L C (P. tetraurelia) C (Sathrophilus sp.) C (T. thermophila) C (Colpidium sp.) L L C (Sathrophilus sp.) C (Sathrophilus sp.) L
1
L, grown on the lettuce medium inoculated with Enterobacter aerogenes (Miyake and Beyer 1973); C, grown on the ciliate in parentheses suspended in SMB. 2 Bangalore strain (Hirshfield et al. 1973). 3 Derived from the ATCC strain 30399 purchased from American Type Culture Collection. 4 Formerly Saprophilus (Corliss 1960). 5 Used only to feed other ciliates.
The ciliates grown on Enterobacter aerogenes in the lettuce medium (L in Table 1) were concentrated by centrifugation, washed by and suspended in SMB-III (1.5 mM NaCl, 0.05 mM KCl, 0.4 mM CaCl2, 0.05 mM MgCl2, 0.05 mM MgSO4, 2 mM Na-phosphate buffer pH 6.8, 2 × 10-3 mM EDTA) (Miyake 1981) (called SMB below), and used after one day. Debris in the culture was removed as described (Miyake and Honda 1976) using a nylon net with a mesh size appropriate to the respective ciliate. Those grown on small ciliates (C in Table 1) were used one day after they had consumed the food. Culture, handling of ciliates and experiments were performed at 24 ± 1°C unless specified.
Bleaching of Stentor The colouration of S. coeruleus was observed with unaided eyes on a white background. This simple method allowed to distinguish between extensively bleached white cells and less-bleached pale-blue cells, both of which looked similar (grayish-brown) in a stereomicroscope with transillumination. Bleaching by high-temperature: Cells grown at temperatures higher than 29 °C gradually lost the bluish colour. At 30°C the bleaching was nearly complete after
Chemical defence in Stentor
10 days, but cells grew more slowly, stopped growing several days later and started dying after several more days. At 29 °C cells turned pale blue after about 10 days and remained so for at least 2 months. Cells continued growing and they looked as healthy as unbleached cells (see also Fig. 1C). Bleaching by caffeine: Following Tartar’s (1972) method, we bleached S. coeruleus by growing cells in the presence of 0.2 mM caffeine (Caffeine anhydrous, Fluka). Cells in the caffeine-containing culture medium multiplied normally except during the first 3–4 days, turned pale blue after several days and remained so until the culture was discontinued after 2 months. Caffeinebleached cells looked as healthy as unbleached cells; they conjugated with unbleached cells of the complementary mating type. Before use, cells were washed 2–3 times by repeating mild centrifugation in SMB and incubated in SMB for 2 hours. Bleaching by lysozyme: Lysozyme, which induces a massive discharge of trichocysts in live cells of Paramecium (Harumoto and Miyake 1991), induced a massive discharge of pigment granules in S. coeruleus. Cells were treated with 50 and 100 µg/ml lysozyme (crystallized lysozyme from hen egg, Boehringer Mannheim) in SMB for 10 minutes, washed with and suspended in SMB as caffeine-treated cells described above. Cells so treated looked pale blue, but otherwise normal (see also Fig. 3C).
Stentorin Chemically-synthesized stentorin (Iio et al. 1995) was dissolved in 75% ethanol (2 mg/ml) and stored in the dark at 2–6 °C. The solution was diluted with SMB at the time of the experiment.
Toxicity-test of stentorin Ten cells of a ciliate were placed in 250 µl of stentorin solution of various concentrations and the number of surviving cells were counted after 24 hours. The LD50 concentration of stentorin for a ciliate was obtained based on the concentration-survival curve of the ciliate (Fig. 4). Unless specified, cells were kept in a dark moist chamber until the time of observation.
Results 1. Observations on the interaction between a Stentor and a Dileptus Dileptus attacks prey with a long flexible toxicysts-bearing proboscis. A single hit with the proboscis can induce an instant total disintegration of small prey like Colpidium (Dragesco 1962) and inflicts a local lysis in large prey such as S. coeruleus
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(Visscher 1923) and Blepharisma japonicum (Miyake et al. 1990). Five cells of Dileptus and 30 cells of Stentor were placed in 250 µl SMB in a depression slide and the outcome of the encounter between a Dileptus and a Stentor was observed in a stereomicroscope. A characteristic interaction occurred when the proboscis of a Dileptus touched a Stentor. If the Stentor was extended, it immediately contracted and, within a second, a mass of bluish material appeared between the Stentor and the proboscis. The Dileptus swam backward often with the bluish material, which was clinging to the proboscis. Sometimes a bluish cloud-like mass was seen rising from the Stentor at or near the site touched by the proboscis. If another Dileptus happened to touch the “blue cloud”, the Dileptus pulled back indicating that the cloud contains a factor which induces backward swimming in Dileptus. Since the Stentor, but not the Dileptus, was blue coloured, it was deduced that the blue cloud contained material derived from the Stentor. The retreated Dileptus often had a less pointed and slightly shorter proboscis and the cell became sluggish. If a Dileptus stayed with a Stentor for several seconds before retreating, these changes were more prominent. In spite of these pathological reactions, a retreated Dileptus sometimes ingested a part or the whole of the bluish material, which the Dileptus carried with its proboscis. In some cases, an attacking Dileptus was surrounded by the blue cloud, sank to the bottom of the container, remained in the cloud and gradually became spherical in the following hour. In most cases, a retreated Dileptus attacked a Stentor again, but as they repeated attacks, the shortening of the proboscis continued. The Dileptus often lost the entire proboscis, became spherical and died in several hours. When other stocks of S. coeruleus and D. margaritifer were used, the result was essentially the same. In D3–I and F1–4 of Dileptus, which are larger than SHL1, the backward swimming and the pathological reactions were slightly less prominent. In Mün 20 and Pol 1 of Stentor, which are smaller but more intensely coloured, the mass of bluish material was more deeply coloured, clung to the proboscis of Dileptus more often and apparently injured Dileptus more severely. These observations indicate that S. coeruleus defends itself against the attack by D. margaritifer and suggest that the blue pigment of S. coeruleus, stentorin, participates in the defence.
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A. Miyake, T. Harumoto and H. Iio
Table 2. Offence-defence interaction between one Stentor (S) and 1, 2, 5, 7 and 10 cells of Dileptus (D) in 100 µl SMB. Mean numbers of surviving cells from 3 experiments. Time
Cells
(day)
1S–1D
1S–2D
1S–5D
1S–7D
1S–10D
S
D
S
D
S
D
S
D
S
D
0
1.0
1.0
1.0
2.0
1.0
5.0
1.0
7.0
1.0
10.0
1
1.0
0.0
0.7
1.0
0.3
3.7
0.0
9.3
0.0
11.3
2
1.0
0.0
0.7
1.0
0.3
4.3
0.0
9.3
0.0
11.6
2. Interaction between a Stentor and various numbers of Dileptus One cell of Stentor was placed with 1, 2, 5, 7 and 10 cells of Dileptus in 100 µl SMB in a depression slide. These cell mixtures, which were called 1S–1D, 1S–2D, 1S–5D, 1S–7D and 1S–10D, respectively, were made in triplicate and observed for 2 days (Table 2). In 1S–7D and 1S–10D, Stentor disappeared and Dileptus slightly increased in number suggesting that Dileptus consumed Stentor as food. On the contrary, in 1S–1D, it was Dileptus that was killed. In 1S–2D and 1S–5D, both Stentor and Dileptus decreased in number probably killing each other. The result indicates that Dileptus can prey on Stentor only when it is numerically superior over Stentor under these conditions, supporting the assumption that Stentor can defend itself against Dileptus. The result also confirms the observation that Stentor can kill Dileptus.
3. Observations on the interaction between a bleached Stentor and a Dileptus Five cells of Dileptus and 30 cells of bleached Stentor were placed in 200 µl SMB in a depression slide and the outcome of the encounter between a Dileptus and a Stentor was observed in a stereomicroscope. When a Dileptus attacked the Stentor, which had been almost completely bleached after growing at 30 °C for 12 days, the Dileptus seldom retreated, immediately started ingesting the Stentor, and continued the predation, while not showing any sign of affliction. A bleached Stentor is thus more vulnerable to the predator than an unbleached one. However, in the Stentor culture kept at 30 °C for
12 days cells were not entirely normal (see Material and Methods), making it difficult to ascribe the observed higher vulnerability to the reduced colouration. If Stentor was only partially bleached by high temperature, caffeine or by lysozyme, cells appeared as healthy as unbleached ones (see Material and Methods). The interaction between such a partially bleached Stentor and a Dileptus was qualitatively the same as the one between an unbleached Stentor and a Dileptus; the Dileptus retreated after attacking the Stentor and looked afflicted. However, both the behavioural and pathological reactions of the Dileptus looked weaker, suggesting that the partially bleached Stentor is more vulnerable to the predator than the unbleached one. This assumption was tested in the following experiments.
4. Interaction between Dileptus and partially bleached Stentor Stentor was partially bleached by 3 different methods, high temperature, caffeine and lysozyme treatment, and compared with unbleached Stentor as prey for Dileptus. High-temperature bleached Stentor Stentor was bleached by growing at 29 °C for 15 days. Cells were pale blue but looked as healthy as unbleached cells. Five cells of bleached and unbleached Stentor were placed with 5 cells of Dileptus in 250 µl SMB in a depression slide in 21 duplicates and observed for 48 hours (Fig. 1A–B). Both the bleached and unbleached Stentor decreased in number, but the decrease was more
Chemical defence in Stentor
extensive in the bleached one (Fig. 1A). After 48 hours, survival rates were 1% and 85% for the bleached and unbleached Stentor, respectively. The bleached Stentor is, therefore, more vulnerable to predation by Dileptus. The Dileptus mixed with unbleached Stentor decreased in number indicating that it met with a strong counter-attack from the Stentor (Fig. 1B). On the contrary, the Dileptus mixed with the bleached Stentor multiplied, indicating that the Dileptus consumed the Stentor as food (Fig. 1B). In parallel with these experiments, the bleached and unbleached cells of Stentor were fed (Fig. 1C). The bleached cells were not slower in growth than the unbleached cells, both dividing about twice in 48 hours, and supporting the observation that the bleached cells were as healthy as unbleached cells. Caffeine-bleached Stentor Stentor was bleached by growing in the presence of caffeine (0.2 mM) for 7 days. Bleached cells were apparently normal except in colouration (Material and Methods). Five cells of bleached and unbleached Stentor, were placed in 250 µl SMB with 10 cells of Dileptus in a depression slide in 21 duplicates and observed for 48 hours (Fig. 2A–B). Bleached Stentor steadily decreased in number while unbleached Stentor changed little in number (Fig. 2A) indicating that the bleached Stentor is more vulnerable to predation by Dileptus.
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The Dileptus with unbleached Stentor decreased in number indicating that the Dileptus met with a strong counter-attack from Stentor. The Dileptus with bleached Stentor decreased a little in number at the beginning but then multiplied, indicating that the Dileptus preyed on the bleached Stentor after an initial suffering from the counter-attack by the Stentor. Lysozyme-bleached Stentor In the first experiment, Stentor was bleached by suspending cells in 50 and 100 µg/ml lysozyme for 10 minutes. In both treatments, bleached cells looked similarly pale blue. Unbleached cells for the control were treated in the same way including the washing by repeated centrifugation (Material and Methods) except that they were suspended for 10 minutes in SMB instead of the lysozyme solution. A Stentor, either bleached or unbleached, was placed with a Dileptus in 50 µl SMB in a depression slide in 20 duplicates and observed for 3 days (Table 3). After 2 days, unbleached Stentor survived in 7 mixtures, while bleached Stentor survived only in 1 mixture. Bleached Stentor was killed in all 20 mixtures in 3 days not killing Dileptus at all, while unbleached Stentor survived in 4 mixtures killing Dileptus in all of them. The bleached Stentor is, therefore, more vulnerable to Dileptus than an unbleached one.
Fig. 1. Effect of partial bleaching of Stentor coeruleus by high temperature (29 °C, 15 days) on the offence-defence interaction between S. coeruleus and Dileptus margaritifer. The number of Stentor and that of Dileptus are plotted in A and B, respectively, against the time after mixing 5 cells of Dileptus with 5 bleached (s–––s)* and 5 unbleached (d–––d)** cells of Stentor. In C, bleached (s–––s)* and unbleached (d–––d)** cells of Stentor were compared for their ability to multiply by feeding 5 cells. Means of 21 experiments with SE. *Solid line, unfilled circles; **Solid line, filled circles.
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Table 3. Effect of partial bleaching of Stentor coeruleus by lysozyme on the offence-defence interaction between S. coeruleus and Dileptus margaritifer in 20 oneto-one mixtures. Stentor
Survived cells Time (day) 0
1
2
3
Unbleached
Stentor Dileptus
20 20
15 20
7 20
4 16
Fig. 2. Effect of partial bleaching of Stentor coeruleus by caffeine (0.2 mM, 7 days) on the offence-defence interaction between S. coeruleus and Dileptus margaritifer. The number of Stentor and that of Dileptus are plotted in A and B, respectively, against the time after mixing 10 cells of Dileptus with 5 bleached (s–––s)* and 5 unbleached (d–––d)** cells of Stentor. Means of 21 experiments with SE. *Solid line, unfilled circles; ** Solid line, filled circles.
Bleached by 50 µg/ml lysozyme
Stentor Dileptus
20 20
7 20
0 20
0 20
Bleached by 100 µg/ml lysozyme
Stentor Dileptus
20 20
10 20
1 20
0 20
Significantly, the Stentor treated with 100 µg/ml lysozyme was not more vulnerable than the one treated with 50 µg/ml, in spite of the fact that the chemically-induced abnormality is usually more extensive at higher concentrations of the inducing agent. Since both treatments were the same in their effect on the bleaching (see above), the result supports the assumption that the higher vulnerability of the bleached Stentor is mainly due to the bleaching.
In the second experiment, Stentor was bleached by suspending cells in 50 µg/ml lysozyme for 10 min. Unbleached Stentor was treated in the same way except that they were suspended for 10 minutes in SMB instead of the lysozyme solution. Lysozyme-treated Stentor was distinctly paler than untreated cells, but looked as healthy as untreated cells. Five cells of either bleached or unbleached Stentor were placed with 5 cells of Dileptus in 250 µl SMB in a depression slide in 21 duplicates and
Fig. 3. Effect of partial bleaching of Stentor coeruleus by lysozyme (50 µg/ml, 10 mins) on the offence-defence interaction between S. coeruleus and Dileptus margaritifer. The number of Stentor and that of Dileptus are plotted in A and B, respectively, against the time after mixing 5 cells of Dileptus with 5 bleached (s–––s)* and 5 unbleached (d–––d)** Stentor cells. In C, bleached (s–––s)* and unbleached (d–––d)** cells of Stentor were compared for their ability to multiply by feeding 5 cells. Means of 21 experiments with SE. *Solid line, unfilled circles; **Solid line, filled circles.
Chemical defence in Stentor
83
Fig. 4. Lethal effect of stentorin on various ciliates. A: Lembadion bullinum. B: Paramecium tetraurelia. C: Dileptus margaritifer stock SHL1. D: Dileptus margaritifer stock D3-I. E: Didinium nasutum. F: Euplotes octocarinatus. G: Stentor polymorphus. H: Stentor coeruleus. I: Blepharisma japonicum. J: Climacostomum virens. Mean numbers of surviving cells of 3 experiments with SE.
observed for 28 hours (Fig. 3A–B). In the mixture of bleached Stentor and Dileptus, Stentor decreased in number (Fig. 3A) while Dileptus increased in number (Fig. 3B), indicating that Dileptus preyed on Stentor. On the contrary, in the mixture of Dileptus and unbleached Stentor, the number of Stentor did not decrease (Fig. 3A) while the number of Dileptus decreased (Fig. 3B), indicating that the Stentor not only defended itself from attack by Dileptus, but also effectively counter-attacked the predator. In parallel with these experiments, bleached and unbleached cells of Stentor were compared in their ability to multiply (Fig. 3C). They were indistin-
guishable, both dividing once in 28 hours, supporting the observation that bleached cells were as healthy as unbleached cells.
5. Toxicity of stentorin to Dileptus margaritifer, Stentor coeruleus and other ciliates Behavioural and pathological responses of Dileptus to stentorin Ten cells of Dileptus were placed in 500 µl SMB containing various concentrations of stentorin in a depression slide and observed in a stereomicroscope. At 5–20 µg/ml of stentorin, all cells immedi-
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A. Miyake, T. Harumoto and H. Iio
ately started swimming backward. At 5 µg/ml, cells became sluggish after about 1 minute of backward swimming and their proboscis started degenerating 1–2 minutes later. At 20 µg/ml, the backward swimming lasted only several seconds. Cells then became sluggish and their proboscis started degenerating within a minute. No striking response was observed at concentrations lower than 1 µg/ml. In stock D3-I, the response of cells to stentorin was similar, but they stopped swimming for a few seconds before starting the backward swimming. These responses of Dileptus to stentorin, the backward swimming, sluggishness and degeneration of the proboscis, are quite similar to the reactions of Dileptus after attacking Stentor (Results 1), suggesting that the behavioural and pathological reactions of Dileptus in the Dileptus-Stentor interaction are evoked by stentorin released by Stentor. More quantitative study on the toxicity of stentorin was carried out on the lethal effect. Lethal effect of stentorin on Dileptus and other ciliates The toxicity of stentorin to D. margaritifer (stocks SHL1 and D3-I) and 8 other ciliates was tested as described in Material and Methods (Fig. 4). The LD50 concentration of stentorin for these ciliates, based on the data in Fig. 4, are listed in Table 4. These values range from 0.1 µg/ml (Lembadion) to >100 µg/ml (Blepharisma, Climacostomum). The LD50 for Stentor coeruleus (90 µg/ml) is about
Table 4. Lethal dose 50% (LD50 ) of stentorin for ciliates. Species and stocks
LD50 of stentorin (µg/ml)
Lembadion bullinum Paramecium tetraurelia Dileptus margaritifer SHL1 Dileptus margaritifer D3-I Didinium nasutum Euplotes octocarinatus Stentor polymorphus Stentor coeruleus Blepharisma japonicum Climacostomum virens1
0.1 0.5 0.6 0.9 1.1 2.3 5.0 90.0 >100.0 >100.0
1 “White” cells lacking the colouration due to symbiotic Chlorella.
100 times higher than the LD50 for Dileptus (0.6 and 0.9 µg/ml). The four heterotrichs tested are all more resistant than other ciliates, a possible explanation of which is given in the Discussion. In Dileptus, stock D3-I is more resistant than stock SHL1 (Fig. 4C–D, Table 4), indicating that there is also intraspecific variation in the sensitivity to stentorin. Effect of light on the toxicity of stentorin Since stentorin is a photodynamic pigment (Yang et al. 1986), we examined the effect of light on the toxicity of stentorin. The LD50 of stentorin for Dileptus (D3-I) was measured in the light and in the dark at the same time. The test in the dark was carried out as described in Material and Methods. The test in the light was carried out in the same way except that Dileptus was kept in a glass moist chamber placed under a 40W cylindrical fluorescence bulb at a distance of 40 cm. The LD50 of stentorin in the dark was about 5 times higher than that in the light (Fig. 5), indicating that photodynamic action participates in the toxicity of stentorin in the light.
Conclusion Fig. 5. Effect of light on the lethal effect of stentorin on Dileptus margaritifer (stock D3-I). s–––s*, L: Experiment in the light. d–––d**, D: Experiment in the dark. Mean numbers of surviving cells of 3 experiments with SE. *Solid line, unfilled circles; **Solid line, filled circles.
Observations on the interaction between Dileptus margaritifer and Stentor coeruleus (Results 1 and 2) show that Dileptus attacks Stentor and that Stentor defends itself against the predator. The observation also suggests that the defence of Stentor
Chemical defence in Stentor
is based on the blue pigment, stentorin. This hypothesis is supported by observations on the interaction between bleached Stentor and Dileptus (Results 3), and more strongly by the experimental result that bleaching of Stentor by three different methods made this ciliate more vulnerable to Dileptus without inducing any detectable abnormalities except in colouration (Results 4). The hypothesis is also supported by the result that chemically-synthesized stentorin induced all the behavioural and pathological responses of Dileptus observed in the Dileptus-Stentor interaction, except the ingestion of and the entanglement with the material released from Stentor (Result 5). In addition, the result that the pigment was about 100 times more toxic to Dileptus than to Stentor is consistent with the hypothesis (Results 5). We, therefore, conclude that stentorin participates in the defence of Stentor coeruleus against the predatory ciliate Dileptus margaritifer. Since stentorin is localized in pigment granules of S. coeruleus, we also conclude that pigment granules of S. coeruleus have the function of defence.
Discussion Function of pigment granules in Stentor coeruleus and other heterotrich ciliates With the above conclusion, this work verifies the old assumption of Tartar (1961) that the pigment in Stentor coeruleus might have some protective value against predators. His assumption was based on 1) the unpublished work of K. M. Møller and A. H. Whiteley (published as appendix in (Møller 1962)) that the alcohol-extracted pigment of some races of Stentor coeruleus was photolethal to Paramecium caudatum and Colpidium, and 2) the speculation that the red pigment of Stentor igneus might be identical to the red pigment of Blepharisma known to be toxic to certain other protozoa (Giese 1949). Møller (1962) considered the possible defence function of pigment granules as a somewhat far-fetched explanation for their function. Giese (1973a) once assumed that the red pigment of Blepharisma might have a defence function against predators, but, having no direct evidence for this function, he thought that the most likely function of the pigment is protection against far UV radiation (Giese 1973a, 1973b, 1981).
85
Later, Estève (1982) compared dozens of ciliates as prey for Dileptus visscheri, Dileptus sp. and Lacrymaria olor and noted that these predators showed “reticence” to ingest pigmented heterotrichs, including S. coeruleus. He interpreted the phenomenon as food selection in predators, but the result could be interpreted also as an indication of the protective function of pigments in these heterotrichs. More recently, the hypothesis on the defence function of pigment granules in B. japonicum was experimentally verified (Miyake et al. 1990; Harumoto et al. 1998), and this prompted us to study the defence function of the pigment in S. coeruleus. Tartar (1961) also thought that stentorin might have a photoreceptor function. This assumption was verified more quickly. It has been shown that stentorin is the photoreceptor pigment for the step-up photophobic response and negative phototaxis of S. coeruleus (Song et al. 1980, see Song 1981; Song et al. 1991 for review). Later, blepharismin was also shown to function as a photoreceptor for the photophobic behaviour of B. japonicum (Scevoli et al. 1987; Matsuoka et al. 1992, 1995; Checcucci et al. 1993; see Ghetti 1991 for review). Thus pigment granules in B. japonicum and S. coeruleus have two functions, defence against predators and photoreception, suggesting that pigment granules in other heterotrichs also have these functions. Harumoto et al. (1998) discussed the two functions in B. japonicum and proposed that the primary function of pigment granules is chemical defence. The same argument can be applied to S. coeruleus. Indeed, some features of pigment granules of S. coeruleus, such as the presence in large numbers throughout the cell surface and the discharge of their content in responding to various external stimuli, conform better to the defence function. Tartar (1961) tested 8 races of S. coeruleus for the negative response to daylight and found that two showed a strong response, two showed no response and the rest showed intermediate response. The result suggests that the photophobic response of S. coeruleus is the subject of a wide variation among stocks. On the other hand, in the present work all of the 4 stocks of S. coeruleus tested strongly repelled the predator. These results are consistent with the assumption that chemical defence is the primary function of pigment granules.
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A. Miyake, T. Harumoto and H. Iio
Relevant to this question is the presence of many colourless or apparently colourless heterotrichs including Stentor polymorphus (Tartar 1961) and Blepharisma hyalinum (Larsen and Nilsson 1983). They have colourless or colourlesslooking cortical vesicles, which are morphologically similar to pigment granules. This raises questions; whether their cortical vesicles contain a minute amount of pigment for photoreception, and whether they contain a colourless toxin for defence. The latter question was examined in recent studies on the heterotrich Climacostomum virens, which has colourless cortical vesicles (Peck et al. 1975). Masaki et al. (1999) isolated a toxic substance from the extract of C. virens, identified it as 1, 3-dihydroxy-5-[(Z)-2′-nonenyl]benzene, named it climacostol and suggested that climacostol serves for defence against predators. It was subsequently demonstrated that cortical vesicles of C. virens function as organelles for chemical defence against the predatory ciliate Dileptus margaritifer and that the chemical basis of this defence is climacostol (Miyake A. Buonanno F. and Saltalamacchia P.; Abstracts, 3rd Eur. Congr. Protistol., 9th Eur. Conf. Ciliate Biol., Helsingør, Denmark, 1999 p.50; personal communication). Assuming that colourless cortical vesicles and pigment granules in heterotrichs are homologous organelles, and hence that pigment granules are pigmented cortical vesicles, this work on C. virens suggests that chemical defence is a general function of cortical vesicles in heterotrichs. Masaki et al. (1999) suggest that the climacostol is biochemically related to stentorin. The possibility that defence toxins in heterotrichs are chemically related to each other, provides a hint to understand the result that the four heterotrichs tested are all more resistant than other ciliates to stentorin (Fig. 4, Table 4).
Defence against predators in Stentor coeruleus S. coeruleus is a large contractile sedentary ciliate. It usually attaches to a substratum with the posterior end and extends itself. During swimming, it is often contracted to some extent. These cells rapidly contract when responding to various stimuli, e.g., an attack by Dileptus margaritifer. Both the large size and the contractility have protective values against the predator. Be-
cause of its size, S. coeruleus can survive the first hit with the proboscis of D. margaritifer, which instantly kills small ciliates like Tetrahymena pyriformis. The large size also makes it possible to leave a piece of destroyed cytoplasm as a bite for the predator to help in escaping. The contraction may serve for separating Stentor from the predator and also from the toxin released by the predator. It may also provide a mechanical stimulus to the predator to induce a retreating reaction. In addition, S. coeruleus can defend itself chemically by means of toxin-containing extrusomes, pigment granules, as shown in this work. Reactions of Dileptus in the Dileptus-Stentor interaction described in this work are backward swimming, slowing down, ingestion of and entanglement with the material released from Stentor, degeneration of the proboscis, pathological rounding of the cell, and death. They are all helpful for Stentor in escaping from the predator. The lethal reaction of Dileptus is probably a laboratory artifact, however. It took several hours under the condition in which Dileptus is confined in a small space with Stentor and, therefore, may not occur in nature where more space is available for dispersion of cells and dilution of toxins. On the other hand, the partial degeneration of the proboscis occurs more quickly and is important in the defence of Stentor against Dileptus in nature. Most important for the defence appears to be the behavioural reaction of Dileptus. The backward swimming of attacking Dileptus provides Stentor a good chance to escape. The slowing down of the retreated predator further facilitates the escape. If the retreated predator ingests a piece of the material released from Stentor or if it is entangled with it, Stentor gains even longer time to escape. Since the retreating and pathological reactions of Dileptus are all induced by chemically synthesized stentorin, we assume that stentorin in pigment granules is responsible for these reactions. The possibility that other unknown toxins also participate in these reactions is not entirely excluded, however. The backward swimming of Dileptus may be induced by the sudden contraction of Stentor, but a fully contracted Stentor also induced the backward swimming indicating that stentorin can induce this reaction without any help of the contraction. On the other hand, the ingestion of and entanglement with the material released from Stentor requires the physical integrity that stentorin alone cannot provide.
Chemical defence in Stentor
Pigment granules in S. coeruleus contain glycoproteins and a lipoprotein (Song 1981). Stentorin in pigment granules associates with glycoproteins to form chromoproteins (Kim et al. 1990), which are thought to function as photoreceptors (Kim et al. 1990; Song et al. 1990). These proteins may serve, together with other components in pigment granules, for maintaining the physical integrity of the discharged material of pigment granules. If so, these proteins play their own role in the defence function of pigment granules. Stentorin has two kinds of toxicity, the phototoxicity, which is light dependent, and the intrinsic toxicity, which is independent of light. Since stentorin is, like blepharismin (Giese 1973a), a photodynamic pigment (Yang et al. 1986), the toxicity of stentorin in the light is partly due to photodynamic action, i.e., photo-oxidation mediated by a photosensitizer pigment. Indeed, stentorin was about 5 times more toxic to D. margaritifer even under moderately illuminated conditions (Results 5, Fig. 5). Since the behavioural and pathological reactions of Dileptus reported in this work were observed under ordinary illumination, both phototoxicity and intrinsic toxicity participated in these reactions. When the Dileptus-Blepharisma interaction was observed under red light, which is scarcely absorbed by blepharismin, the reactions of Dileptus were virtually the same as those observed under ordinary illumination, indicating that the intrinsic toxicity of blepharismin alone can induce these reactions (Harumoto et al. 1998). Since stentorin is chemically closely related to blepharismin and since the pigment is toxic in the dark (Results 5), it is inferred that the intrinsic toxicity of stentorin can induce the behavioural reactions of Dileptus. The intrinsic toxicity of stentorin makes it possible to maintain the defence function at night. Even in daytime, it is probably important for the defence, as S. coeruleus tends to stay in shady places because of the photophobic behaviour. Little is known about the molecular mechanism of the intrinsic toxicity of stentorin. The problem of how pigment granules are discharged in the Dileptus-Stentor interaction can be inferred, based on the study on the DileptusBlepharisma interaction. Stereomicroscopic observations on the Dileptus-Stentor interaction indicate that the discharge of pigment by Stentor is localized at and near the site touched by the proboscis of Dileptus. This is quite similar to that which occurs in the Blepharisma-Dileptus inter-
87
action. In the latter, observations in a scanning electron microscope at the moment of the attack by Dileptus (Harumoto et al. 1998) showed that pigment granules of Blepharisma were discharged at the area adjacent to the site, which was physically damaged by the attack. At this area, the cell surface was morphologically intact except for the presence of many round depressions, each of which was thought to have been produced by the discharge of a pigment granule. The discharge of pigment granules occurs, therefore, at the intact cell surface as a response to the stimulus coming from the attack by Dileptus. The discharge of pigment granules in S. coeruleus attacked by Dileptus probably occurs in the same way. Interestingly, S. coeruleus is apparently defenceless against the predatory ciliate Spathidium sp. (Kuhlmann H.-W. personal communication, our unpublished observation). Studies on the interaction between these two ciliates will provide new insight for understanding the defence mechanism of S. coeruleus against predators. Acknowledgement: We thank Dr. M. Tavrovskaya, Inst. Cytol. Russ. Acad. Sci., St. Petersburg, for the strain D3-I of Dileptus margaritifer, Dr. K. Golinska, Nencki Inst. Exp. Biol., Warsaw, for strains L and SH of D. margaritifer, Drs. K. Heckmann and H.-W. Kuhlmann, Inst. Gen. Zool. & Genet., Univ. Münster, for help in collecting Stentor stocks, and Ms. V. Rivola and Mr. P. Saltalamacchia, Univ. Camerino, for technical assistance. This work was supported by Italian CNR and MURST.
References Cameron D. W. and Riches A. G. (1995): Synthesis of stentorin. Tetrahedron Lett. 36, 2331–2334. Checcucci G., Damato G., Ghetti F. and Lenci F. (1993): Action spectra of the photophobic response of the blue and red forms of Blepharisma japonicum. Photochem. Photobiol. 57, 686–689. Corliss J. O. (1960): The problem of homonyms among generic names of ciliated protozoa, with proposal of several new names. J. Protozool. 7, 269–278. Dragesco J. (1962): Capture et ingestion des proies chez les infusoires ciliés. Bull. Biol. Fr. Belg. 96, 123–167. Dragesco J. (1984): Organites sous-cuticulaires éjectables, éjectisomes (trichocystes). In: Grassé P.-P. (ed.): Traité de zoologie II. Infusoires ciliés I. pp.181–199. Masson, Paris. Estève J.-C. (1982): Défaillance de controle du alimentaire chez les ciliés gymnostomes Dileptus et Lacrymaria. Protistologica 18, 517–526. Giese A. C. (1949): A cytotoxin from Blepharisma. Biol. Bull. 97, 145–149.
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