The behavioural responses to light in the ciliate Paramecium bursaria Focke, which normally contains hundreds of the symbiotic green alga Chlorella in its cytoplasm, were analysed quantitatively to clarify the mechanisms governing photoreception in the cell. P. bursana was found to possess three kinds of photoreceptor systems for (1) the step-up photophobic response (system I), (2) the step-down photophobic response (system II), and (3) the photokinetic response (system III). Under the influence of light, the symbiotic algae inhibited systems I and HI, but activated system II. Thus the cells showed the avoiding reaction when they encountered shade (the step-down photophobic response), and consequently gathered in the light region (photoaccumulation). Inhibition of system I and activation of system II were assumed to be mediated by products of the blue-light effect of the algae, while inhibition of system III was due to photosynthetic products of the algae. The cells whose algae were experimentally removed gathered in the shade (photodispersal) due to the avoiding reaction exhibited by them when they encountered a lighted region (the step-up photophobic response mediated by system I). Lowered swimming velocity and increased frequency of spontaneous changes in the swimming direction in the shade (photokinetic responses mediated by system III) also caused photodispersal.
The ciliate protozoan Paramecium bursaria contains hundreds of green algae, Chlorella, in its cytoplasm. Engelmann (1882) found that when a specimen of P. bursaria swimming in a light region encountered shade it exhibited an avoiding reaction (step-down photophobic response; Diehn el al. 1977) and returned to the light region (photoaccumulation; Diehn et al. 1977; Diehn, 1979; Jennings, 1906).
Iwatsuki & Naitoh (1981) demonstrated that a potent inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-l’,l-dimethylurea (DCMU), in the external solution had no effects on either the step-down photophobic response or photoaccumulation in P. bursana. They also found that the spectral sensitivity curve for the photophobic response was different in its major peak wavelength (520 nm) from those for the photosynthetic activity of the algae (440nm and 680nm). The ineffectiveness of DCMU in inhibiting the photophobic response was also reported by Cronkite & Van Den Brink (1981).
In contrast, Niess, Reisser & Wiessner (1981, 1982) reported that DCMU caused a loss of the photophobic response of P. bursaria, and the peak wavelength of the spectral sensitivity curve for the response coincided with that of the action spectrum for photosynthesis in the green algae. However, Pado (1972) reported that blue light (450 nm) was most effective in inducing photoaccumulation in P. bursaria.
Photoaccumulation of microorganisms is dependent on both the phobic response exhibited by the organisms at the light-dark border and their kinetic activities, such as forward swimming velocity (orthokinesis) and the frequency of spontaneous change in swimming direction (klinokinesis). Photoaccumulation is also influenced by light-direction-oriented locomotion of the organisms (phototaxis) (Diehn, 1979).
To understand the mechanisms governing the photobehaviour of P. bursaria in relation to its symbiotic green algae, we examined quantitatively the photophobic responses, the photokinetic responses, and phototaxis in both Chlorella-containing and Chlorella-free specimens. Based on these results, we hypothesize that the specimens of P. bursaria possess at least three kinds of photoreceptor systems responsible for (1) the step-down photophobic response, (2) the step-up photophobic response and (3) the photokinetic responses, respectively. Since symbiotic Chlorella modify the systems through their photosynthesis and blue-light effect (non-photosynthetic carbon metabolism; Kamiya & Miyachi, 1974; Miyachi, Miyachi & Kamiya, 1978; Senger, 1980), they change the photobehaviour of their host. Some of the discrepancies between the results obtained by previous workers can then be explained.
MATERIALS AND METHODS
Chlorella-free specimens of Paramecium bursaria were obtained by keeping the normal Chlorella-containing specimens (syngen 1, mating type IV) in complete darkness for more than 60 days (Muscatine, Karakashian & Karakashian, 1967). Both Chlorella-containing and Chlorella-free specimens were cultured in a bacterized wheat straw infusion at 21 ± 1°C under a fixed illumination cycle of 12 h dark and 12h light (3Wm−2 fluorescent lamps). Specimens obtained from culture 3h after the light had been turned on were used as the light-adapted specimens. Those obtained from culture kept in the dark for 3 h more after the end of the dark period were used as the dark-adapted specimens (Iwatsuki & Naitoh, 1978, 1979, 1981). All specimens were washed gently with a standard saline solution for 2-5 min prior to experimentation (Naitoh & Eckert, 1972).
About 2 ml of the suspension of equilibrated cells was introduced into a cylindrical Plexiglas dish (30 mm inner diameter) under illumination with a dim light (background light; 50mWm−2) for observation and videorecording of specimen locomotion. A stimulating light was applied to the specimens through the bottom of the vessel. Light intensity was suddenly increased from the background level to a higher stimulation level (step-up photostimulation), and kept at that level for 2 min. Light intensity was then suddenly decreased to the original background level (stepdown photostimulation). To indicate the degree of the response, the number of specimens which showed a photophobic response to photostimulation was counted on a replayed picture, and presented as a percentage of the total number of specimens (40-60) in the dish.
Monochromatic light was obtained by putting an interference filter (halfbandwidth; less than 18 nm) and a cut-off filter (Toshiba) in front of a light source (a 650-W halogen lamp). Light intensity was controlled by neutral density filters and monitored with a photometer (Spectra PR-1000). The thermal radiation from the light source was minimized by an infrared cut-off filter. All the experiments were performed at 21 ± 2°C. Some details of the experimental methods will appear in the Results.
Photophobic responses in the Chlorella-free specimens
Some specimens in a single population of the dark-adapted Chlore lla-free P. bursaria showed the step-up photophobic response, whereas others showed the step-down photophobic response. The number of specimens that showed each response increased with increasing stimulus intensity, up to a maximum (Fig. 1A). When the intensity was more than 2 W m−2, about 70 % of the specimens showed the step-up response, while the remaining 30% showed the step-down response. The threshold intensities for the half-maximal response (35% step-up and 15% stepdown) were about 0-20 W m”2 and 0-26 W m−2, respectively.
Long-term (10 min) observation of a single specimen stimulated every 30 s by a white light of 2Wm−2 revealed that it showed both the step-up response and the step-down response. About 70% of the responses during this time were step-up, while 30% were step-down. The light-adapated Chlorella-free specimens showed photophobic responses identical with the dark-adapted ones.
Photophobic responses in the Chlorella-containing specimens
Photophobic responses exhibited by the dark-adapted Chlorella-containing specimens were identical to those that were Chlorella-free, i.e. 70% of the specimens showed the step-up and 30 % the step-down response when exposed to a supramaximal photostimulation (2 W m∼2). In contrast, the light-adapted Chlorella-containing specimens showed only the step-down photophobic response. The number of specimens which showed the response increased with increasing stimulus intensity. All showed the response when light intensity was over 2Wm 2 (Fig. IB). The threshold for the half-maximal response (50%) was about 0·09 W m−2.
Action spectra for the photophobic response
To find the most effective light for inducing photophobic responses, we determined the relationship between the degree of response and the intensity of the stimulating light (fluence rate) using nine monochromatic lights of different wavelengths, ranging from 400 to 720 nm. Two examples (520 nm and 600 nm) of the ‘fluence rate-response curves’ are shown in Fig. 2. The degree of response increased almost linearly with a logarithmic increase in the fluence rate, reaching a maximum. This was about 70 % for the step-up response (Fig. 2A) and about 30 % for the stepdown response (Fig. 2B) in the Chlorella-free specimens. It was 100% for the stepdown response in light-adapted Chlorella-containing specimens (Fig. 2C).
The fluence rate corresponding to the half-maximal response was estimated for each curve. The reciprocal of the rate (the relative response, which corresponds to the effectiveness of the light in inducing the response) was plotted against the wavelength to obtain the action spectrum for the response. As shown in Fig. 3A, the action spectrum for the step-up response showed a major peak at 560 nm and a minor peak at 680 nm, while that for the step-down response showed a single peak at 520 nm in the Chlorella-free specimens. Action spectra for both step-up and step-down responses in the dark-adapted Chlorella-containing specimens were identical to those in the Chlorella-free specimens (Fig. 3B). The action spectrum for the step-down response in the light-adapted Chlorella-containing specimens showed a single peak at 520nm (Fig. 3C).
The light-induced change in the photophobic responses in the dark-adapted chlorella-containing specimens
As described in the previous section, about 70% of the dark-adapted Chlorella- containing specimens showed the step-up photophobic response, and the rest showed the step-down photophobic response. When the dark-adapted specimens were kept exposed to a white light (light adaptation), all eventually showed only a step-down response. To measure the time courses for suppression of the step-up response and enhancement of the step-down response by light, the dark-adapted Chlorella- containing specimens in the culture were exposed to a white light (3Wm−2) for varying periods, then the photophobic responses of the specimens were examined. A 520 nm monochromatic light with an intensity of 4x 1018 quanta m−2s−1 was used for photostimulation. The intensity of the background light for the videorecording was 2x 1016quantam−2s−1. As shown in Fig. 4, the step-up photophobic response decreased from its original level (67 %) to zero, while that for step-down increased from its original level (13 %) to 100% with the time of exposure.
To examine the involvement of photosynthesis by the symbiotic Chlorella in the light-induced changes in photophobic responses, the time courses for light-induced changes were determined in the presence of 10 ‘moll−1 DCMU, an inhibitor of photosynthesis, in the external solution. The time courses were identical with those obtained in the absence of DCMU (Fig. 4).
Spectral sensitivity curves for the light-induced changes in the photophobic responses
To find the most effective light for suppressing the step-up response and enhancing the step-down response in the dark-adapted Chlorella-containing specimens, we subjected them to various monochromatic lights of different wavelengths (400-720nm; 4X1018quantam−2s−1) for 2h, then examined their photophobic responses. A 520 nm monochromatic light with an intensity of 4X1018 quanta m−2s−1 was used for stimulation. The degree of suppression or enhancement was defined as the difference between the response of dark-adapted specimens and those subjected to each monochromatic light for 2h, and was plotted against the wavelength to obtain the spectral sensitivity curve. The curves shown in Fig. 5 clearly indicate that 480 nm light was the most effective for both suppression and enhancement.
The photophobic response to a change in light intensity subsided in about 15 s. As that response declined, forward swimming velocity and the frequency of spontaneous changes in swimming direction (kinetic activities) reached their respective steady levels, which depended on the light intensity, in about 1 min. These levels remained unchanged for 5-30 min until the specimens showed thigmotaxis (the thigmotactic specimens did not swim but crept slowly on the bottom of their container: Iwatsuki & Naitoh, 1979). Kinetic activities were therefore measured 1 min after the light intensity had been changed.
When the light intensity was increased to more than 50 Wm−2, the swimming velocity of the Chlorella-free. specimens increased (positive photo-orthokinetic response), reaching a maximum (about l−9mms−1; 130% of the swimming velocity in the dark), whereas the frequency of spontaneous change in the swimming direction decreased (negative photoklinokinetic response), reaching its minimum of about 0·02Hz (25% of the frequency in the dark) (Fig. 6A). The threshold intensities for half-maximal responses were 2·1 Wm−2 for the orthokinetic response and 0·75 Wm−2 for the klinokinetic response. The dark-adapted Chlorella-contain- ing specimens showed photokinetic responses identical to those of the Chlorella-tree specimens.
In light-adapted Chlorella-containing specimens, however, photokinetic responses were inconspicuous. That is, in light of intensity greater than 40 Wm−2, swimming velocity increased by less than 5 % and the frequency of spontaneous changes in the swimming direction decreased by less than 20% of each value in the dark (Fig. 6B).
The velocity was always higher and the frequency always lower in light-adapted Chlorella -containing specimens than in those that were dark-adapted (or Chlorella- free) when they were put in the dark (0-0·05 W m−2). In the light (50Wm−2), however, maximum velocity was significantly higher in light-adapted specimens than in dark-adapted ones.
Action spectra for the photokinetic responses
To find the most effective light for inducing photokinetic responses in Chlorella- free specimens, the fluence rate-response curves for photokinetic responses were determined with nine different monochromatic lights (400 — 720nm). Two examples of the curves (for 440 and 680 nm) are shown in Fig. 7. Each kinetic activity changed quasilinearly with a logarithmic increase in the fluence rate to reach its maximum. The fluence rate of each monochromatic light corresponding to the half-maximal kinetic response (the orthokinesis, 1·7 mm; the klinokinesis, 0·049 Hz) was determined. The reciprocal of the fluence rate (the relative response) was plotted against the wavelength to obtain the action spectrum. The action spectrum for the orthokinetic response and that for the klinokinetic response showed a single peak at 440 nm (Fig. 8). The action spectra obtained for Chlorella-free. and for dark-adapted Chlorella -containing specimens were similar.
Light-induced suppression of the photokinetic responses in dark-adapted Chlorellacontaining specimens
As described above, the photokinetic responses in the dark-adapted Chlorella- containing specimens became inconspicuous after light exposure (Fig. 6B). To determine the time course for light-induced suppression of photokinetic responses, the degree of each photokinetic response of dark-adapted specimens was determined after exposing them to white light (5Wm∼2) for various times (0—120min). The degree of the response, defined here as the difference between the kinetic activity of dark-adapted specimens in background light (520nm: 2X1017quantam−2s−1) and stimulating light (520nm: 4X1018quantam−2s−1, strong enough to produce the maximal response), is presented as a percentage of the maximum degree seen in the specimens before their exposure to white light (at 0 exposure time). The photokinetic response decreased exponentially with the exposure time, reaching a minimum in about 120 min (Fig. 9).
To examine whether the photosynthetic activity of the symbiotic green algae is involved in the mechanism of light-induced suppression, the time course for suppression was determined in the presence of 10−5moll−1 DCMU in the external solution. The suppression at 12 min was less than 10 %, in contrast to more than 90 % in the absence of DCMU (Fig. 9).
Spectral sensitivity curves for light-induced suppression of the photokinetic responses
To find the most effective light for suppressing photokinetic responses, the darkadapted C/z/ore//a-containing specimens were exposed to various monochromatic lights (4x 1018quantam−2s−1) with different wavelengths (400-720nm) for 120min, then the percentage of each photokinetic response was determined. The difference between this value and 100% (an indication of the degree of the suppression) was plotted against each corresponding wavelength to obtain the spectral sensitivity curve for suppression. Suppression of the orthokinetic response and the klinokinetic response occurred most effectively at 440 and 680 nm (Fig. 10).
Examination of the phototaxis
To determine whether specimens ofP. bursaria show phototaxis, we examined the swimming direction of about 400 specimens in an experimental vessel subjected to both collimated and diffused white light (l0Wm−2).
The swimming direction of a specimen was defined as the angle between the direction of the collimated light and the direction to which the anterior end of the specimen pointed. There was no significant directional difference in movement between specimens in collimated light and diffused light, or between Chlorella-tree specimens and light-adapted Chlorella -containing specimens (Fig. 11).
Behavioural responses to light in dark-adapted Chlorella-containing specimens of P. bursaria were identical with those that were Chlorella-free. All showed the avoiding reaction to an increase in light intensity (step-up photophobic response) and accumulated in the dark region (photodispersal). Forward swimming velocity (orthokinesis) was higher and the frequency of spontaneous changes in swimming direction (klinokinesis) was lower when the specimens were in the lighter region.
Higher swimming velocity and a lower frequency of changes in swimming direction in the lighter region decrease the probability that the specimens will stay in the region. Therefore, photokinetic responses (changes in the kinetic activities) also cause photodispersal.
However, light-adapted Chlorella-containing specimens showed an avoiding reaction to a decrease in light intensity (step-down photophobic response), and thus they accumulated in the light region (photoaccumulation). The kinetic responses in the light-adapted specimens were weak, and thus the responses did not interfere with the photoaccumulation mediated by the step-down photophobic response.
It is interesting to note that both Chlorella-free. and dark-adapted Chlorella- containing specimens showed not only step-up photophobic but also step-down photophobic responses, though the former was predominant (Iwatsuki & Naitoh, 1981). The peak wavelengths of the action spectrum differed between the two photophobic responses (560 nm and 680 nm for the step-up, 520 nm for the stepdown; Fig. 3A). This suggests that the two photophobic responses are under the control of two different photoreceptor systems. They will be termed ‘photoreceptor system I’ for the step-up response and ‘photoreceptor system II’ for the step-down response. Nevertheless, the action spectra for the two kinetic responses showed a single peak at a common wavelength of 440 nm (Fig. 8). This implies that the two photokinetic responses are under the control of a single photoreceptor system, ‘photoreceptor system III’.
Under the influence of light, the symbiotic Chlorella inhibits activities of systems I and III, but enhances that of system II (Figs 3, 6). Thus the specimens show photoaccumulation. Clearly, photoaccumulation of the host is essential for survival of the photosynthetic symbionts. Some of the photosynthetic products are known to benefit the host (Brown & Nielsen, 1974).
The symbiont-mediated, light-induced suppression of system I and the enhancement of system If were not inhibited by DCMU, an inhibitor of photosynthesis (Fig. 4). Moreover, the spectral sensitivity curves for both suppression and enhancement showed a single peak at a common wavelength, 480 nm. Interestingly, this peak does not correspond to any of the photosynthetic action spectrum peaks but does correspond to the blue-light effect of the symbionts (Kamiya & Miyachi, 1974; Miyachi et al. 1978). These results suggest that both suppression and enhancement are mediated by the blue-light effect of the symbionts. However, DCMU inhibited the suppression of system III (Fig. 9). Moreover, the two peaks of the spectral sensitivity curves for the suppression coincided with those of the action spectrum for the photosynthesis of symbionts (440 and 680 nm; Fig. 10) (Haxo, 1960). This may mean that the suppression is mediated by the photosynthesis of symbionts. The long lag period for suppression or enhancement (Figs 4, 9; 60-90min) suggests that at least some products of either the blue-light effect or photosynthesis must accumulate before such responses occur.
The photophobic response is caused by a transient reversal in the beat direction of cilia (ciliary reversal). This reversal is due to an inflow of Ca2+ into the cilia through the depolarization-activated Ca2+ channels in the ciliary membrane (Eckert, Naitoh & Machemer, 1976; Naitoh, 1982). It is conceivable, therefore, that light stimulation of photoreceptor systems I and II produces a transient depolarizing receptor potential, which spreads electrotonically to the ciliary membrane to activate the Ca2+ channels. A sustained membrane hyperpolarization causes a sustained increase in the forward swimming velocity, due primarily to an increase in ciliary beat frequency, and it suppresses the spontaneous avoiding reaction in Paramecium (Machemer, 1976; Naitoh, 1982). Photoreceptor system III, therefore, may produce prolonged membrane hyperpolarization in response to a sustained increase in light intensity. Our preliminary (unpublished) examinations of Chlorella-free specimens reveal that a transient membrane depolarization followed by a sustained hyperpolarization occurred during increasing light intensity. These hypothetical receptor systems of P. bursaria and their modifications by the symbiotic Chlorella are presented schematically in Fig. 12.
Photoaccumulation is dependent on both the step-down photophobic response exhibited by the specimens at the light—dark border and on the photokinetic responses. If the rate of decrease in light intensity experienced by specimens at a light—dark border is below a certain threshold (Saji & Oosawa, 1974) the specimens do not show the photophobic response at the border. Their accumulation, therefore, depends mostly on kinetic responses. However, if the rate of decrease is large enough to produce a photophobic response, accumulation may depend primarily on the phobic response. The contribution from each response to photoaccumulation may vary according to light intensity, light intensity gradient at the light—dark border, shape and size of the experimental vessel, ionic composition of the experimental solution, temperature, culture age, circadian time, nutritive condition, etc. We controlled carefully for these factors. In our experimental system, photoaccumulation was dependent almost exclusively on the photophobic response (Iwatsuki & Naitoh, 1981, 1983). The administration of DCMU did not affect photoaccumulation, since DCMU did not affect the photophobic response (Fig. 4). If, for some reason, the phobic response at the border is weak, suppression of kinetic responses is indispensable for accumulation that is mediated by the phobic response. This is because such responses (i.e. increased forward swimming velocity and decreased frequency of the spontaneous light-avoiding reaction) antagonize photoaccumulation. Since suppression of the photokinetic response was photosynthesis-dependent, inhibition of the symbiont’s photosynthesis by DCMU abolished photoaccumulation, as in the cases reported by Niess el al. (1981, 1982). Interaction between the photophobic response and motile activity of the cells whilst accumulating in the light was clearly demonstrated by Cronkite & Van Den Brink (1981). They found that light-induced (supposedly O2-mediated) suppression of motile activity in creeping Chlorella-containing specimens of P. bursaria was a cause of photoaccumulation of the specimens. Administration of DCMU removed the suppression, and the specimens became motile. However, the motile specimens stayed in a light spot, because they showed the step-down photophobic response at the light—dark border even in the presence of DCMU.
Our finding, that blue light inhibited the step-up photophobic response and enhanced the step-down photophobic response, is consistent with that of Pado (1972), i.e. dark-adapted specimens accumulated in the blue-light region of spectral components of a white light. The specimens that entered the blue-light region showed a step-down photophobic response and thus stayed in the region. Further detailed examinations of the photoresponses are needed to explain some discrepancies between our results and those of Reisser & Haeder (1984).
Common colourless Paramecium, such as P. caudatum, P. tetraurelia (Iwatsuki & Naitoh, 1982) and P. multimicronucleatum (Iwatsuki & Naitoh, 1983) showed a stepup photophobic response and resulting photodispersal in a manner similar to Chlorella-íree P. bursaria. However, these colourless Paramecium, which have never established a symbiotic relationship with Chlorella, did not show the stepdown photophobic response seen in Chlorella-free P. bursaria. It is conceivable that the establishment of the symbiotic relationship of P. bursaria with the green algae is dependent on their possession of photoreceptor system II, which mediates the stepdown photophobic response indispensable for photoaccumulation.
This work was supported by grants from the Ministry of Education of Japan (59480019), Mitsubishi Foundation and from Nippon Petrochemicals Company. We are grateful to our colleagues, Professor T. Ikawa for his valuable suggestions and Professor J. B. Bowles for his critical reading of the manuscript.