The defense function of pigment granules in the red ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus and Climacostomum virens, was investigated by (1) comparing normally-pigmented and albino mutant cells of B. japonicum as the prey of these predators and (2) comparing resistance of the predators to blepharismin, the toxic pigment contained in the pigment granules of B. japonicum. Normally pigmented cells which contained more blepharismin than albino cells were less vulnerable to A. proteus than albino cells, but not to C. virens. C. virens was more resistant than A. proteus to the lethal effect of blepharismin. The results indicate that pigment granules of B. japonicum function as defense organelles against A. proteus but not against C. virens and suggest that successful defense against a predator depends on the susceptibility of the predator to blepharismin.
The pigment granules of Blepharisma japonicum are membrane-bounded spherical organelles 0.3–0.6 μm in diameter that are mostly localized in the cortex and attached to the cell membrane (Inaba et al., 1958; Giese, 1973; Jenkins, 1973). They contain the red photodynamic pigment, blepharismin (Checcucci et al., 1997; Maeda et al., 1997), and the contents of the granules are discharged from the cell in response to various chemical and physical stimuli (Giese, 1973). The granules are, therefore, considered to be extrusomes or extrusive organelles in protists (Hausmann, 1978).
Two functions of pigment granules in B. japonicum have been experimentally demonstrated: (1) chemical defense against the predatory ciliate Dileptus margaritifer (Miyake et al., 1990; Harumoto et al, 1998; Terazima et al., 1999) and (2) photoreception for photophobic responses (Matsuoka et al., 1992; Checcucci et al., 1993). In addition, Giese (1973, 1980) showed that pigment granules serve as a protective shield against far UV radiation. The biological significance of this function was questioned, however, because this range of solar radiation scarcely reaches the surface of the present-day earth (Harumoto et al., 1998). The chemical basis of these functions is the red pigment, blepharismin.
The evidence for the chemical defense function of the pigment granules is as follows. The albino mutant and bleached cells of B. japonicum are much more vulnerable to the predator (Miyake et al., 1990). B. japonicum discharges pigment granules at and near the attacked site when it is attacked by D. margaritifer (Harumoto et al., 1998). Purified blepharismin is highly toxic to the predator, in the light as well as in the dark, while the pigment is almost non-toxic to B. japonicum (Harumoto et al., 1998; Terazima et al., 1999). Oxyblepharismin, the photoinduced product of blepharismin, is also toxic to the predator (Terazima et al., 1999).
In this work we investigated whether pigment granules in B. japonicum are effective in defense against two other predatory protists, Amoeba proteus, a rhizopod, and Climacostomum virens, a ciliate, by comparing normally-pigmented and albino mutant cells of B. japonicum as prey for these predators and by measuring the susceptibility of these predators to purified blepharismin. The result showed that pigment granules of B. japonicum are effective in the defense against A. proteus, which is sensitive to blepharismin, but that they are not effective against C. virens, which is resistant to blepharismin.
MATERIALS AND METHODS
Blepharisma japonicum, stocks R1072 (wild-type) and A538 (mutant albino), Climacostomum virens, clone W-24, isolated by Dr. M. Tavrovskaya, Inst. Cytol., Russ. Acad. Sci., St. Petersburg, and Amoeba proteus, stock G, provided by Dr. Y. Tsukii, Hosei Univ., Tokyo, were used.
The mutant albino of B. japonicum appeared spontaneously in the laboratory among wild-type red cells (Chunosoff et al., 1965). It contains only a minute amount of the red pigment blepharismin and looks white (Chunosoff et al., 1965; Giese and Grainger, 1970). For more details about stocks of Blepharisma, see Harumoto et al., 1998. C. virens is usually green because of the presence of symbiotic Chlorella. A permanently white subclone (clone W) was isolated from the clone W-24 and used in this work.
Blepharisma was grown in the dark at 25°C in the wheat-grass-powder medium (Takagi et al., 1993) inoculated with Enterobacter aerogenes 2 days before use. Cells were collected by centrifugation (100×g, 3 min), washed with SMB-(1.5 mM NaCl, 0.05 mM KCl, 0.4 mM CaCl2, 0.05 mM MgCl2, 0.05 mM MgSO4, 2.0 mM Na-phosphate buffer, pH 6.8), a modified medium of SMB, which is a synthetic medium for Blepharisma (Miyake et al., 1981), filtered through a nylon net to remove debris, resuspended in SMB-, and used after 1 day. Climacostomum and Amoeba were grown on SMB-suspension of Sathrophilus sp., a small ciliate, that was grown, washed and suspended in SMB-as Blepharisma. Only slightly starved cells in the stationary phase were used for the experiments. Handling of cells and experiments were performed at room temperature (23±5°C). Experimental cells were kept in dark, moist chambers except during handling and observation.
Five predators (Climacostomum or Amoeba) were placed in 200 μl SMB- with 10, 20, and 40 blepharismas. These mixtures were made for albino and red blepharismas. In each mixture, the numbers of living blepharismas and predators were counted each day for 4 days. Data are the means of nine experiments.
Blepharismin was extracted and purified as indicated elsewhere (Terazima et al., 1999). Blepharismin was dissolved in 99.5% ethanol and further diluted with SMB- so that the final concentration of ethanol was less than 2% (vol/vol). The concentration of blepharismin was calculated based on the molar extinction coefficient of blepharismin in ethanol, 3.75×104 M−1cm−1 (Checcucci et al., 1997).
Lethal effect of blepharismin
The lethal effect of blepharismin on a protist was tested by placing ten cells of the protist in 200 μl of SMB-containing various concentrations of blepharismin in a slide depression and by counting the number of living cells after 1 day. The lethal dose 50% (LD50) of blepharismin was obtained using the concentration-survival curve as described elsewhere (Harumoto et al., 1998).
To test the defensive function of pigment granules in red Blepharisma, we compared normally pigmented and albino mutant cells of B. japonicum in the interaction with amoebas. In the mixtures containing albino blepharismas, the number of blepharismas decreased (Fig. 1, A), while the number of amoebas increased (Fig. 1, a). Amoebas multiplied more intensively in the mixture in which more blepharismas disappeared, indicating that albino blepharismas were consumed as food by amoebas. On the contrary, normally-pigmented red blepharismas survived. In the mixtures containing red blepharismas, the number of blepharismas decreased only slightly in 5A-10B and did not decrease at all in 5A-20B and 5A-40B (Fig. 1, B). The number of amoebas did not change (Fig. 1, b). They were often floating just under the surface of the medium, and the phenomenon was not observed in the mixture containing albino blepharismas, suggesting that amoebas were affected by red blepharismas but not by albino blepharismas.
Albino blepharismas are, therefore, much more vulnerable to amoebas than red blepharismas, indicating that blepharismin, and hence also pigment granules which contain the pigment, have a defense function against Amoeba proteus.
Climacostomum virens has a large mouth opening. Preys of various sizes are sucked into the buccal cavity and ingested through the cytostome. Blepharismas, albino and red, were mixed with climacostoma in 200 μl SMB- in a slide depression, and the interaction between the two protists was observed in a stereomicroscope. Climacostoma ingested both albino and red blepharismas apparently indiscriminately. Albino and red blepharismas were compared more quantitatively as prey for climacostoma (Fig. 2). In all of the interactions in any of the mixtures, the number of blepharismas decreased (Fig. 2, A, B) while the number of climacostoma increased (Fig. 2, a, b). In 5C-10B and 5C-20B, albino and red blepharismas decreased in number in nearly the same way. In 5C-40B, more red cells survived after 1 day, but the difference largely disappeared after 2 days. The slightly higher increase in the number of climacostoma in the mixtures with red blepharismas is probably due to the fact that red blepharismas are slightly larger than albino blepharismas. The result indicates that albino and red blepharismas were similarly consumed as food by climacostoma. Albino and red blepharismas are, therefore, equally vulnerable to climacostoma, indicating that pigment granules of B. japonicum are not effective for the defense against C. virens.
Resistance to blepharismin
To investigate further the role of blepharismin in the interactions between the protists used in this work, the resistance of these protists to blepharismin expressed by LD50 was compared, the concentration of blepharismin required for 50% lethality (Table 1). The resistance of Blepharisma (red, albino) was so high that LD50 was not measurable at the concentrations tested. Climacostomum, against which blepharismin was not effective in the defense, was 29 times more resistant than Dileptus, against which blepharismin is known to be effective in the defense (Miyake et al., 1990). Amoeba, against which blepharismin was effective in the defense, was only twice as resistant as Dileptus. These results suggest that the defense function of blepharismin against predatory protists is based on the susceptibility of these predators.
Lethal dose 50% (LD50) of blepharismin in the dark for the four species of protists used in this work.
The purified blepharismin was highly toxic to various ciliates (Harumoto et al, 1998), suggesting that the defensive function of red B. japonicum is widely effective against various predatory ciliates. This finding was not verified in the cell-cell interaction, however, except in the case of the Blepharisma-Dileptus interaction (Miyake et al., 1990). So we studied the defensive function of the red B. japonicum against heterotrophic heterotrich C. virens, which is different from D. margaritfer in its mode of feeding.
On the other hand, the observation that rhizopod Actinosphaerium eichhorni ingests many red blepharismas suggests that pigment granules have no protective function against this predator (Giese, 1973). We examined the defensive function of pigment granules in the red B. japonicum against the predatory protist Amoeba proteus, which belongs to the same phylum Sarcomastigophora as Actinosphaerium eichhorni. Amoeba is different in its mode of feeding from D. margaritifer and C. virens. Whether the defensive function of the pigment granules of B. japonicum is effective against the other predatory protists A. proteus and C. virens or not is a question that attracts much interest.
Our results show that normally pigmented albino cells of B. japonicum are more vulnerable than red cells to the predatory rhizopod A. proteus but not to the predatory ciliate C. virens. Since the red coloration of B. japonicum is due to the pigment, blepharismin, localized in pigment granules (Inaba et al., 1958; Giese, 1973), the result indicates that pigment granules of B. japonicum function as defense organelles against A. proteus but not against C. virens. These facts suggest that the pigment granules in B. japonicum are effective not only against D. margaritifer but also other predatory protists, and are not effective against some others.
Our finding that C. virens is much more resistant than D. margaritifer and A. proteus to the lethal effect of blepharismin (Table 1) suggests that the successful defense by pigment granules against a predatory protist is due to a high susceptibility of the predator to blepharismin.
On the other hand, in the defense of B. japonicum against D. margaritifer, pigment granules discharge blepharismin in response to the predator's attack (Harumoto et al., 1998), while the pigment is not discharged when blepharismas are ingested by Actinosphaerium (Giese, 1973), suggesting that the successful defense by pigment granules against a predatory protist depends on the capacity of blepharismas to discharge pigment granules as a response to the predator's attack. To confirm these assumptions, further studies, particularly the study of the discharge of pigment granules of B. japonicum at the time of attack by A. proteus and C. virens, and the study of the susceptibility of A. eichhorni to blepharismin, are needed.
Blepharismin is a photodynamic pigment. Even a dilute solution of blepharismin photosensitizes colorless cells (Giese, 1953; Giese, 1973; Harumoto et al., 1998). The toxicity of blepharismin to D. margaritifer is much higher under illuminated conditions (Harumoto et al., 1998; Terazima et al., 1999). When Blepharisma is exposed to strong light in the presence of oxygen, it is killed by photodynamic action due to its own pigment blepharismin (Giese and Zeuthen, 1948; Giese, 1953). Up to now, the demonstration of the defense function of blepharismin was mostly based on experiments carried out in the dark (Miyake et al., 1990; Harumoto et al., 1998; this work). The intrinsic toxicity (the toxicity in the dark) of blepharismin is, therefore, sufficient for the use of this pigment as a chemical defense, but to understand fully the mechanism of defense by means of blepharismin, it is necessary to consider also the toxicity due to the photodynamic action against both Blepharisma and its predator.
The mechanism of the defense function of pigment granules in the light is made more complicated by the fact that light changes blepharismin to oxyblepharismin (Giese and Zeuthen, 1948; Giese and Grainger, 1970; Ghetti et al., 1992; Watanabe et al., 1995: Spitzner et al., 1998). We found that oxyblepharismin also has intrinsic toxicity and phototoxicity and that it also plays a role in the defense function of the pigment granules in B. japonicum (Terazima et al., 1999). The phototoxicity of blepharismin is thought to be due to the generation of short-lived oxygen species, such as singlet oxygen (1O2)(Jardon et al., 1987; Checcucci et al., 1991) and hydroxyl radical (OH ·)(Kato et al., 1996). Very little is known about the mechanism of the intrinsic toxicity of blepharismin (Pant et al., 1997).
Some heterotrich ciliates other than B. japonicum have pigment granules. Of these, Stentor coeruleus is known to exhibit chemical defense against D. margaritifer by means of the pigment stentorin, which is localized in the pigment granules of this ciliate (Harumoto and Miyake, 1996; Miyake et al., 2001).
Many other heterotrichs, such as C. virens (Peck et al., 1975) and Blepharisma hyalinum (Larsen and Nilsson, 1983), have colorless cortical vesicles which are morphologically similar to pigment granules. The cortical vesicles in C. virens were recently shown to be extrusomes that have a defensive function against D. margaritifer (Miyake et al., 2003). That research also showed that the chemical basis of this defense is climacostol discharged from cortical vesicles. Climacostol is the colorless toxin isolated from the whole extract of C. virens by Masaki et al. (1999), who suggested that climacostol is metabolically related to stentorin and hypericin from plants, and hence is probably also related to blepharismin. These findings suggest that the study of the chemical evolution of extrusomal defense toxins in ciliates has interesting possibilities.
We are grateful to Dr.Y.Takagi of Nara Women's Univ. and Dr.H.Iio of Osaka City Univ. for helpful discussion.