Journal of Photochemistry
and Photobiology,
B: Biology,
5 (1990)
105 - 114
105
EFFECTS OF UV RADIATION ON MOTILITY, PHOTO-ORIENTATION AND PIGMENTATION IN A FRESHWATER Cryptomonas DONAT-P. HADERT and MARIA H;iDER Znstitut fiir Botanik und Pharmazewtische Staudtstr. 5, D-8520 Erlangen (F.R.G.)
Biologie der Friedrich-Alexander-Uniuersitiit,
(Received March 7, 1989; accepted July 11, 1989)
Keywords. Absorption spectra, Cryptomonas, image analysis, motility, photobleaching, phototaxis, UV radiation, videomicroscopy.
Summary The effects of UV radiation on photo-orientation, motility and pigmentation have been studied in a freshwater Cryptomonas species. The cells show a pronounced diaphototactic orientation which is affected by UV radiation at 50 mW m-* within about 90 min. Both the average velocity of the swimming cells and the percentage of motile cells within the population decrease within about the same exposure time. UV radiation also bleaches the cellular pigments.
1. Introduction Orientation with respect to a number of relevant external stimuli is used by many motile phytoplankton species to optimize their position in the water column [l, 21. Light is an essential requirement for a photosynthetic organism; therefore an upward movement directed by positive phototaxis or another mechanism is an ecological necessity for many flagellates [3 - 61. However, an unrestricted upward movement would lead the organisms to the surface where they would be exposed to unfiltered solar radiation; many micro-organisms cannot tolerate this unfiltered radiation which causes photobleaching and eventual damage of the cells [7 - 91. Unattenuated solar radiation irreversibly damages colourless and photosynthetic organisms within a few hours [lo - 121. The UV component of radiation has been found to be the most inhibitory since removal of short wavelengths by inserting appropriate filters increases the tolerated exposure times [ 12 - 141. TAuthor to whom correspondence should be addressed. loll-1344/90/$3.50
@ Elsevier Sequoia/Printed
in The Netherlands
106
Cryptophycean algae are among the most productive phytoplankton organisms. They form algal blooms both in the oceans and in freshwater habitats which underlines their global importance in the biological food web [ 15, 161. Different species seem to rely on different orientation strategies to find a suitable position in the water column. A freshwater Cryptomonas has been reported to utilize exclusively positive phototaxis at all light intensities [17, 181. In contrast, the marine Cryptomonas maculutu uses a pronounced negative phototaxis at fluence rates of 15 W m-* or greater and a less obvious positive phototaxis at lower fluence rates [19]. The photoreceptor has not been identified in any Cryptomonas species. The action spectrum for phototaxis extends from about 400 nm to 680 nm with a main maximum at 560 nm which corresponds to the absorption of phycoerythrin, the main accessory pigment in this organism [ 18,201. The photosynthetic electron transport chain does not seem to be the coupling site for the photoresponse, since the chlorophylls a and c are not represented in the action spectrum. In addition, the inhibitors 3-(3’,4’-dichlorophenyl)-l,l-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which impair the non-cyclic electron transport chain, do not affect phototaxis [ 191. Recently, phototaxis has been studied in another freshwater Cryptomonas species which orients itself by swimming diaphototactically (perpendicular) to the light beam [21, 221. Thus the cells move in a horizontal plane. This basic strategy is modulated since at low fluence rates the cells deviate slightly upward from the horizontal plane and at high fluence rates slightly downward from the horizontal plane which causes the population to move up or down gradually. These organisms also show a pronounced negative gravitaxis [ 221. Results obtained with specific quenchers and diagnostic reagents suggest that the high fluence response (but not the low fluence photo-orientation) can be mediated by a photodynamic reaction of type II which involves singlet oxygen ( ‘02) production, since 1,4-diazabicyclo[2.2.2]octane (DABCO) and other singlet oxygen scavengers affect this response. In contrast, p-quinone, a free radical quencher, has no effect on the response at any fluence rate [ 211. There is an indication for a periodic shading mechanism in Cryptomonas, unlike the green flagellate Euglena, for which the shading hypothesis has been shown not to apply [23,24]. Repetitive light pulses induce the same phototactic orientation in Cryptomonas as continuous light if the dark interval between flashes does not exceed half the rotation time [25]. Adding a high viscosity medium increases the rotation time and also prolongs the permitted dark interval between flashes [ 261. Removal of Ca*+ suppresses phototactic orientation, but motility is not impaired [27], indicating that the sensory transduction chain involves a Ca*+-dependent step as in a number of other micro-organisms [ 28 - 311. The aim of this paper is to study and quantify the effects of UV radiation on motility, photo-orientation and pigmentation in a freshwater Cryptomonas species.
107 2. Materials and methods 2.1. Organisms and culture conditions An unidentified Cryptomonas species, isolated from a pond near Marburg, was used for all experiments described here. Cultures were grown in Erlenmeyer flasks containing 40 ml of a medium described in ref. 21 at 20 “C. A light-dark cycle was applied consisting of 14 h at 1.3 W rnp2from mixed fluorescence lamps followed by 10 h of darkness. 2.2. UV radiation The cells were exposed in a square open glass cuvette (50 mm X 50 mm, filled with a layer (3 mm) of the cell suspension, 2 X lo5 cells ml-‘) under an inverted transilluminator (Bachofen, Reutlingen, F.R.G.). Light was filtered using a quartz neutral density filter of the reflective type which transmits 13%. The radiation was about 50 mW rnp2 at any given wavelength in the UV-B band, determined using an Optronics (Optronics, Orlando, FA, model 742) double monochromator spectroradiometer. 2.3. Measurements of motility and photo-orientation The photo-orientation of a population was assayed at regular time intervals in samples taken from populations during UV exposure. The direction of movement was determined in individual cells picked at random using an automatic image analysis system [ 321. The samples were allowed to swim in a glass cuvette (inner dimensions, 40 mm X 8 mm X 0.17 mm) placed on the stage of a dark field microscope (Olympus, model BH-2). A CCD camera (Philips LDH 0600) mounted on top of the microscope recorded the image of the cells using an IR monitoring beam from the built-in light source in combination with a 715 nm cut-off filter. IR radiation was chosen so that the photo-orientation was not disturbed (phototactically- or photosynthetically-active radiation would have disturbed the photo-orientation). The signal from the CCD camera was digitized at a spatial resolution of 512 X 512 pixels with 255 grey levels each with a digitizer board (PIP-1024, Matrox, Quebec, Canada) which was plugged into an IBM AT-compatible microcomputer (TCS-7000, Tatung, Teipei, Taiwan). The analysis program was developed in Assembler language in order to allow real time analysis [ 32, 331. Subsequent programs calculated histograms from the raw data and performed a fast Fourier analysis [ 341. Phototaxis was induced by white actinic light (5 W mv2) produced from a 250 W quartz halogen slide projector (Prado, Leitz, Wetzlar, F.R.G.). The percentage of motile organisms and the individual velocities were assayed using the same hardware. The positions of individual cells were determined in images about 330 ms apart. From the centres of gravity at the beginning and end of the time interval, the distance travelled could be measured and the time interval was read from the built-in hardware clock of the computer. Subsequent programs calculated the percentage of motile
108
organisms in the population as well as histograms of the velocity distribution. 2.4. Measurements of absorp tion spectra Cell suspensions were placed in quartz spectrophotometer cuvettes with an optical path length of 10 mm. The suspensions were prevented from settling by stabilizing the organisms in 0.5% agar prepared with growth medium. The cuvettes were filled free of bubbles and were closed with lids. Absorption spectra were measured before (control) and at regular time intervals during UV radiation in a single beam spectrophotometer (Beckman, DU 70); they were transferred into the memory of a connected IBM ATcompatible microcomputer. The initial control spectrum was subtracted from all the other spectra. Thus the decrease in the absorption spectra at the various wavelength band(s) reflects the bleaching of individual pigments. Specific plotting, smoothing and adjusting routines were written in Pascal (Turbo Pascal, Borland International).
3. Results The cells showed a pronounced diaphototactic orientation in a test light beam of 20 klx before exposure to UV radiation (Fig. l(a)). After an exposure time of 20 min the degree of orientation decreased noticeably (Fig. l(b)). When exposed for 40 min (Fig. l(c)) and 60 min (Fig. l(d)) the orientation was almost totally lost and the histograms showed a random distribution. After 80 min the number of motile cells in the population
00’
(b) Fig. 1. (continued)
270”
00’
100"
109
270’
so’
(d)
(cl
(e)
2700
80’
180’
1000'
Fig. 1. Effects of UV radiation on photo-orientation of a freshwater Cryptomonas exposed for (a) 0 min, (b) 20 min, (c) 40 min, (d) 60 min and (e) 80 min.
species
decreased drastically so that the histogram showed only a few tracks (Fig. l(e)). The decreasing orientation was quantified by calculating the percentage of cells moving in sectors which deviated by no more than +30” or ?45” from the direction perpendicular to the light beam. Both calculations showed a dramatic decrease in the orientation within a short exposure time; after about 80 min the cells had completely lost their phototactic behaviour (Fig. 2).
110
0
10
20
30
70
40 50 60 WOOurn time [mitt]
90
90
Fig. 2. Percentage of motile cells moving in sector deviating by no more than ?r30” (open circles) or 545” (filled circles) from a direction perpendicular to the incident light beam plotted as a function of the UV exposure time.
The percentage of motile cells in the population started to decrease shortly after the onset of UV radiation (Fig. 3). After 100 min hardly any motile cells could be found in the population. UV radiation also affected the velocity of the cells during the exposure time. Before exposure the cells moved with velocities which varied between 0 and 50 I.tm s-i (Fig. 4). It is interesting to note that UV radiation induced a weak but significant increase in velocity up to exposure times of 60 min (photokinesis). After 60 min the histograms showed a steady decline in the linear speed of movement and after 100 min of radiation most cells were immotile. When a cell suspension was exposed to UV radiation, a dramatic colour change was detected even by visual inspection after short exposure times.
100
-I
‘\._
0 1
0
10
20
I
I
30
40
I
I
50 60 Exposure time [ min
I
I
I
70
90
90
_ I
100
]
Fig. 3. Percentage of motile cells in samples of a freshwater Cryptomonas species taken from populations exposed to UV radiation us. exposure time. Cells moving slower than 20 pm s? are regarded as immotile.
111
L 20
40
60 Exgosun
so
100
120
tlnm[mln]
Fig. 4. Effect of time of exposure to UV radiation on the speed of movement in a freshwater Cryptomonas species.
The absorption spectra determined at regular time intervals after UV radiation showed a gradual decline in the concentration of all photosynthetic pigments, including the phycobilins, carotenoids and chlorophylls (Fig. 5). This decrease was quantified by calculating the absorption at the characteristic wavelengths of specific pigments. The absorption changes were plotted as a function of the exposure time. Not all the pigments were bleached with the same kinetics (Fig. 6). The phycobilins were lost first with a half-life of 4 h, followed by the carotenoids (half-life of 37 h); chlorophyll a pigments were most stable and were bleached with a half-life of 59 h. 4. Discussion The inhibition of photo-orientation and motility in the freshwater Cryptomonas species cannot be attributed to overheating since the transilluminator emits negligible amounts of visible and IR radiation. Thus in contrast with the effects observed on exposure to solar radiation, the inhibition is exclusively due to UV radiation. This is important since typical photobleaching has only been studied by exposing cells to strong visible radiation [ 91. The mechanisms of UV inhibition of photo-orientation and motility are not known. However, DNA does not seem to be the primary target for UV-B
-0.600
350
400
450
500
550
Wavelength
600
650
700
i
0
[nm]
Fig. 5. Absorption spectra of a freshwater Cryptomonas species measured after increasing times of exposure to LJV radiation (from top to bottom: after 0, 145, 350, 755, 1560, 2915, 3840, 4380, 5615, 6355, 7760, 9205, 10605, 11685, 12 775, 13 960, 14 630, 16 320,17 175 and 18 990 min). llxpoaure time 50
0.
r-0
100
6 I ---o*-_o_ou-oo-~-~_~o-o-
150
1 o-
o-
[h] 200
I o-o-o-_o-oq-•-•-_0-~-0-0--
250
300
# o-o-
; -0
Q-’
>
-0.1
,s -0.2 ‘i b B a -0.3
+\+ -0.4
L+ _ ‘+ +-+-+_+
-+-+_+_
Fig. 6. Decrease in absorption at key wavelengths (representing the major photosynthetic pigments of a freshwater Cryptomonas species) due to UV bleaching after increasing times of exposure: 0,680 nm; 0,480 nm; 0,560 nm; 0,460 nm; +, 440 nm.
113
inhibition in some micro-organisms since first effects can be seen after exposure times of only a few minutes [ 10,121.In addition, photodynamic effects can be eliminated as mechanisms of inhibition by UV radiation since quenchers and scavengers of free radicals and singlet oxygen are not able to remove the inhibition in cyanobacteria and in some green and colourless flagellates [lo, 12,141. Therefore it has been speculated that an intrinsic component of the photoreceptor array and the propulsive machinery of the cell may be the targets of UV radiation causing inhibition of photo-orientation and motility respectively. The sequence of events during bleaching by UV radiation clearly demonstrates that the accessory photosynthetic pigments (phycobilins) are destroyed first, followed by the carotenoids which are believed to play a major role in photoprotection; the chlorophylls essential for photosynthesis are most resistant to UV radiation.
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (SFB 305). The authors gratefully acknowledge the skilful assistance of H. Kropfhauser, R. Krzyzanowsky, J. Schafer, K. Schmidt, D. Schnappinger, S. Seeler and B. Vollertsen. References 1 W. Nultsch and D.-P. HZder, Photomovement in motile microorganisms II, Photothem. Photobiol., 47 (1988) 837 - 869. 2 D.-P. Hiider, Ecological consequences of photomovement in microorganisms, J. Photochem. Photobiol. B, 1 (1988) 385 - 414. 3 R. Hirschberg and W. Hutchinson, Effect of chlorpromazine on phototactic behavior in Chlamydomonas, Can. J. Microbial., 26 (1980) 265 - 267. 4 D.-P. Hider, G. Colombetti, F. Lenci and M. Quaglia, Phototaxis in the flagellates, Euglena gracilis and Ochromonas danica, Arch. Microbial., 130 (1981) 78 - 82. 5 T. Ristori, C. Ascoli, R. Banchetti, P. Parrini and D. Petracchi, Localization of photoreceptor and active membrane in the green alga Haematococcus pluvialis, in Progress in Z’rotozoology, Abstracts, 6th Znt. Congress of Protozoology, Warsaw, Poland, July 5 - 11, 1981, p. 314. 6 R. Wayne, S. Roux and G. Thompson, Photomovement in Dunaliella, in M. Furuya (ed.), Phytochrome and Photomorphogenesis, Proc. 16th Yamada Conf., Okazaki, Japan, October 13 - 17, 1986, p. 103. 7 D.-P. Hader, E. Rhiel and W. Wehrmeyer, Ecological consequences of photomovement and photobleaching in the marine flagellate Cryptomonas maculata, FEMS Microbial. Ecol., 53 (1988) 9 - 18. 8 N. Ekelund and D.-P. Hader, Photomovement and photobleaching in two Gyrodinium species, Plant Cell Physiol., 29 (1988) 1109 - 1114. 9 W. Nultsch and G. Agel, Fluence rate and wavelength dependence of photobleaching in the cyanobacterium Anabaena variabilis, Arch. Microbial., 144 (1986) 268 - 271. 10 D.-P. Hiider, M. Watanabe and M. Furuya, Inhibition of motility in the cyanobacterium, Phormidium uncinatum, by solar and monochromatic UV irradiation, Plant Cell Physiol., 27 (1986) 887 - 894.
114 11 D.-P. Hiider, Effects of solar and artificial UV irradiation on motility and phototaxis in the flagellate, Euglena gracilis, Photochem. Photobiol., 44 (1986) 651 - 656. 12 D.-P. Hgder and M. Hider, Effects of solar UV-B irradiation on photomovement and motility in photosynthetic and colorless flagellates, Environ. Exp. Bot., 29 (1989) 273 - 282. 13 D.-P. Hiider and M. HCder, Ultraviolet-B inhibition of motility in green and dark bleached Euglenagracilis, Curr. Microbial., 17 (1988) 215 - 220. 14 D.-P. Hiider and M. A. Hiider, Inhibition of motility and phototaxis in the green flagellate, Euglena gracilis, by UV-B radiation, Arch. Microbial, 150 (1988) 20 - 25. 15 K. Tangen, Blooms of Gyrodinium aureolum (Dinophyceae) in north European water, accompanied by mortality in marine organisms, Sarsia, 63 (1977) 123 - 133. 16 D. L. Spector, Dinoflagellates, Academic Press, Orlando, FA, 1984. 17 M. Watanabe and M. Furuya, Action spectrum of phototaxis in a cryptomonad alga, Cryptomonas sp., Plant Cell Physiol., 15 (1974) 413 - 420. 18 M. Watanabe, Y. Miyoshi and M. Furuya, Phototaxis in Cryptomonas sp. under condition suppressing photosynthesis, Plant Cell Physiol., 17 (1976) 683 - 690. 19 D.-P. Hader, E. Rhiel and W. Wehrmeyer, Phototaxis in the marine flagellate Cryptomonas maculata, J. Photochem. Photobiol. B, 1 (1987) 115 - 122. 20 M. Watanabe and M. Furuya, Phototactic behavior of individual cells of Cryptomonas sp. in response to continuous and intermittent light stimuli, Photochem. Photobiol., 35 (1982) 559 - 563. 21 E. Rhiel, D.-P. Hiider and W. Wehrmeyer, Photo-orientation in a freshwater Cryptomonas species, J. Photochem. Photobiol. B, 2 (1988) 123 - 132. 22 E. Rhiel, D.-P. Hiider and W. Wehrmeyer, Diaphototaxis and gravitaxis in a freshwater Cryptomonas, Plant Cell Physiol., 29 (1988) 755 - 760. 23 D.-P. Hiider, M. Lebert and M. R. DiLena, New evidence for the mechanism of phototactic orientation of Euglena gracilis, Curr. Microbiob, 14 (1986) 157 - 163. 24 D.-P. Hiider, Polarotaxis, gravitaxis and vertical phototaxis in the green flagellate, Euglenagracilis, Arch. Microbial., 147 (1987) 179 - 183. 25 M. Watanabe and M. Furuya, Phototactic responses of cell population to repeated pulses of yellow light in a phytoflagellate Cryptomonas sp., Plant Physiol., 61 (1978) 816 - 818. 26 H. Uematsu-Kaneda and M. Furuya, Effects of viscosity on phototactic movement and period of cell rotation in Cryptomonas sp., Physiol. Plant., 56 (1982) 194 - 198. 27 H. Uematsu-Kaneda and M. Furuya, Effects of calcium and potassium ions on phototaxis in Cryptomonas, Plant Cell Physiol., 23 (1982) 1377 - 1382. 28 D.-P. Hgder, Gated ion fluxes involved in photophobic responses of the blue-green alga, Phormidium uncinatum, Arch. Microbial., 131 (1982) 77 - 80. 29 D.-P. Hiider and K. L. Poff, Dependence of the photophobic response of the bluegreen alga Phormidium uncinatum on cations, Arch. Microbial., 132 (1982) 345 348. 30 R. Kamiya and G. B. Witman, Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas, J. Cell Biol., 98 (1984) 97 - 107. 31 W. Nultsch, J. Pfau and R. Dolle, Effects of calcium channel blockers on phototaxis and motility of Chlamydomonas reinhardtii, Arch. Microbial., 144 (1986) 393 397. 32 D.-P. Hjider and M. Lebert, Real time computer-controlled tracking of motile microorganisms, Photochem. Photobiol., 42 (1985) 509 - 514. 33 D.-P. Hiider and K. Griebenow, Orientation of the green flagellate, Euglena gracilis, in a vertical column of water, FEMS Microbial. Ecol., 53 (1988) 159 - 167. 34 D.-P. Hgder and E. Lipson, Fourier analysis of angular distributions for motile microorganisms, Photochem. Photobiol., 44 (1986) 657 - 663.
and Photobiology,
B: Biology,
5 (1990)
105 - 114
105
EFFECTS OF UV RADIATION ON MOTILITY, PHOTO-ORIENTATION AND PIGMENTATION IN A FRESHWATER Cryptomonas DONAT-P. HADERT and MARIA H;iDER Znstitut fiir Botanik und Pharmazewtische Staudtstr. 5, D-8520 Erlangen (F.R.G.)
Biologie der Friedrich-Alexander-Uniuersitiit,
(Received March 7, 1989; accepted July 11, 1989)
Keywords. Absorption spectra, Cryptomonas, image analysis, motility, photobleaching, phototaxis, UV radiation, videomicroscopy.
Summary The effects of UV radiation on photo-orientation, motility and pigmentation have been studied in a freshwater Cryptomonas species. The cells show a pronounced diaphototactic orientation which is affected by UV radiation at 50 mW m-* within about 90 min. Both the average velocity of the swimming cells and the percentage of motile cells within the population decrease within about the same exposure time. UV radiation also bleaches the cellular pigments.
1. Introduction Orientation with respect to a number of relevant external stimuli is used by many motile phytoplankton species to optimize their position in the water column [l, 21. Light is an essential requirement for a photosynthetic organism; therefore an upward movement directed by positive phototaxis or another mechanism is an ecological necessity for many flagellates [3 - 61. However, an unrestricted upward movement would lead the organisms to the surface where they would be exposed to unfiltered solar radiation; many micro-organisms cannot tolerate this unfiltered radiation which causes photobleaching and eventual damage of the cells [7 - 91. Unattenuated solar radiation irreversibly damages colourless and photosynthetic organisms within a few hours [lo - 121. The UV component of radiation has been found to be the most inhibitory since removal of short wavelengths by inserting appropriate filters increases the tolerated exposure times [ 12 - 141. TAuthor to whom correspondence should be addressed. loll-1344/90/$3.50
@ Elsevier Sequoia/Printed
in The Netherlands
106
Cryptophycean algae are among the most productive phytoplankton organisms. They form algal blooms both in the oceans and in freshwater habitats which underlines their global importance in the biological food web [ 15, 161. Different species seem to rely on different orientation strategies to find a suitable position in the water column. A freshwater Cryptomonas has been reported to utilize exclusively positive phototaxis at all light intensities [17, 181. In contrast, the marine Cryptomonas maculutu uses a pronounced negative phototaxis at fluence rates of 15 W m-* or greater and a less obvious positive phototaxis at lower fluence rates [19]. The photoreceptor has not been identified in any Cryptomonas species. The action spectrum for phototaxis extends from about 400 nm to 680 nm with a main maximum at 560 nm which corresponds to the absorption of phycoerythrin, the main accessory pigment in this organism [ 18,201. The photosynthetic electron transport chain does not seem to be the coupling site for the photoresponse, since the chlorophylls a and c are not represented in the action spectrum. In addition, the inhibitors 3-(3’,4’-dichlorophenyl)-l,l-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which impair the non-cyclic electron transport chain, do not affect phototaxis [ 191. Recently, phototaxis has been studied in another freshwater Cryptomonas species which orients itself by swimming diaphototactically (perpendicular) to the light beam [21, 221. Thus the cells move in a horizontal plane. This basic strategy is modulated since at low fluence rates the cells deviate slightly upward from the horizontal plane and at high fluence rates slightly downward from the horizontal plane which causes the population to move up or down gradually. These organisms also show a pronounced negative gravitaxis [ 221. Results obtained with specific quenchers and diagnostic reagents suggest that the high fluence response (but not the low fluence photo-orientation) can be mediated by a photodynamic reaction of type II which involves singlet oxygen ( ‘02) production, since 1,4-diazabicyclo[2.2.2]octane (DABCO) and other singlet oxygen scavengers affect this response. In contrast, p-quinone, a free radical quencher, has no effect on the response at any fluence rate [ 211. There is an indication for a periodic shading mechanism in Cryptomonas, unlike the green flagellate Euglena, for which the shading hypothesis has been shown not to apply [23,24]. Repetitive light pulses induce the same phototactic orientation in Cryptomonas as continuous light if the dark interval between flashes does not exceed half the rotation time [25]. Adding a high viscosity medium increases the rotation time and also prolongs the permitted dark interval between flashes [ 261. Removal of Ca*+ suppresses phototactic orientation, but motility is not impaired [27], indicating that the sensory transduction chain involves a Ca*+-dependent step as in a number of other micro-organisms [ 28 - 311. The aim of this paper is to study and quantify the effects of UV radiation on motility, photo-orientation and pigmentation in a freshwater Cryptomonas species.
107 2. Materials and methods 2.1. Organisms and culture conditions An unidentified Cryptomonas species, isolated from a pond near Marburg, was used for all experiments described here. Cultures were grown in Erlenmeyer flasks containing 40 ml of a medium described in ref. 21 at 20 “C. A light-dark cycle was applied consisting of 14 h at 1.3 W rnp2from mixed fluorescence lamps followed by 10 h of darkness. 2.2. UV radiation The cells were exposed in a square open glass cuvette (50 mm X 50 mm, filled with a layer (3 mm) of the cell suspension, 2 X lo5 cells ml-‘) under an inverted transilluminator (Bachofen, Reutlingen, F.R.G.). Light was filtered using a quartz neutral density filter of the reflective type which transmits 13%. The radiation was about 50 mW rnp2 at any given wavelength in the UV-B band, determined using an Optronics (Optronics, Orlando, FA, model 742) double monochromator spectroradiometer. 2.3. Measurements of motility and photo-orientation The photo-orientation of a population was assayed at regular time intervals in samples taken from populations during UV exposure. The direction of movement was determined in individual cells picked at random using an automatic image analysis system [ 321. The samples were allowed to swim in a glass cuvette (inner dimensions, 40 mm X 8 mm X 0.17 mm) placed on the stage of a dark field microscope (Olympus, model BH-2). A CCD camera (Philips LDH 0600) mounted on top of the microscope recorded the image of the cells using an IR monitoring beam from the built-in light source in combination with a 715 nm cut-off filter. IR radiation was chosen so that the photo-orientation was not disturbed (phototactically- or photosynthetically-active radiation would have disturbed the photo-orientation). The signal from the CCD camera was digitized at a spatial resolution of 512 X 512 pixels with 255 grey levels each with a digitizer board (PIP-1024, Matrox, Quebec, Canada) which was plugged into an IBM AT-compatible microcomputer (TCS-7000, Tatung, Teipei, Taiwan). The analysis program was developed in Assembler language in order to allow real time analysis [ 32, 331. Subsequent programs calculated histograms from the raw data and performed a fast Fourier analysis [ 341. Phototaxis was induced by white actinic light (5 W mv2) produced from a 250 W quartz halogen slide projector (Prado, Leitz, Wetzlar, F.R.G.). The percentage of motile organisms and the individual velocities were assayed using the same hardware. The positions of individual cells were determined in images about 330 ms apart. From the centres of gravity at the beginning and end of the time interval, the distance travelled could be measured and the time interval was read from the built-in hardware clock of the computer. Subsequent programs calculated the percentage of motile
108
organisms in the population as well as histograms of the velocity distribution. 2.4. Measurements of absorp tion spectra Cell suspensions were placed in quartz spectrophotometer cuvettes with an optical path length of 10 mm. The suspensions were prevented from settling by stabilizing the organisms in 0.5% agar prepared with growth medium. The cuvettes were filled free of bubbles and were closed with lids. Absorption spectra were measured before (control) and at regular time intervals during UV radiation in a single beam spectrophotometer (Beckman, DU 70); they were transferred into the memory of a connected IBM ATcompatible microcomputer. The initial control spectrum was subtracted from all the other spectra. Thus the decrease in the absorption spectra at the various wavelength band(s) reflects the bleaching of individual pigments. Specific plotting, smoothing and adjusting routines were written in Pascal (Turbo Pascal, Borland International).
3. Results The cells showed a pronounced diaphototactic orientation in a test light beam of 20 klx before exposure to UV radiation (Fig. l(a)). After an exposure time of 20 min the degree of orientation decreased noticeably (Fig. l(b)). When exposed for 40 min (Fig. l(c)) and 60 min (Fig. l(d)) the orientation was almost totally lost and the histograms showed a random distribution. After 80 min the number of motile cells in the population
00’
(b) Fig. 1. (continued)
270”
00’
100"
109
270’
so’
(d)
(cl
(e)
2700
80’
180’
1000'
Fig. 1. Effects of UV radiation on photo-orientation of a freshwater Cryptomonas exposed for (a) 0 min, (b) 20 min, (c) 40 min, (d) 60 min and (e) 80 min.
species
decreased drastically so that the histogram showed only a few tracks (Fig. l(e)). The decreasing orientation was quantified by calculating the percentage of cells moving in sectors which deviated by no more than +30” or ?45” from the direction perpendicular to the light beam. Both calculations showed a dramatic decrease in the orientation within a short exposure time; after about 80 min the cells had completely lost their phototactic behaviour (Fig. 2).
110
0
10
20
30
70
40 50 60 WOOurn time [mitt]
90
90
Fig. 2. Percentage of motile cells moving in sector deviating by no more than ?r30” (open circles) or 545” (filled circles) from a direction perpendicular to the incident light beam plotted as a function of the UV exposure time.
The percentage of motile cells in the population started to decrease shortly after the onset of UV radiation (Fig. 3). After 100 min hardly any motile cells could be found in the population. UV radiation also affected the velocity of the cells during the exposure time. Before exposure the cells moved with velocities which varied between 0 and 50 I.tm s-i (Fig. 4). It is interesting to note that UV radiation induced a weak but significant increase in velocity up to exposure times of 60 min (photokinesis). After 60 min the histograms showed a steady decline in the linear speed of movement and after 100 min of radiation most cells were immotile. When a cell suspension was exposed to UV radiation, a dramatic colour change was detected even by visual inspection after short exposure times.
100
-I
‘\._
0 1
0
10
20
I
I
30
40
I
I
50 60 Exposure time [ min
I
I
I
70
90
90
_ I
100
]
Fig. 3. Percentage of motile cells in samples of a freshwater Cryptomonas species taken from populations exposed to UV radiation us. exposure time. Cells moving slower than 20 pm s? are regarded as immotile.
111
L 20
40
60 Exgosun
so
100
120
tlnm[mln]
Fig. 4. Effect of time of exposure to UV radiation on the speed of movement in a freshwater Cryptomonas species.
The absorption spectra determined at regular time intervals after UV radiation showed a gradual decline in the concentration of all photosynthetic pigments, including the phycobilins, carotenoids and chlorophylls (Fig. 5). This decrease was quantified by calculating the absorption at the characteristic wavelengths of specific pigments. The absorption changes were plotted as a function of the exposure time. Not all the pigments were bleached with the same kinetics (Fig. 6). The phycobilins were lost first with a half-life of 4 h, followed by the carotenoids (half-life of 37 h); chlorophyll a pigments were most stable and were bleached with a half-life of 59 h. 4. Discussion The inhibition of photo-orientation and motility in the freshwater Cryptomonas species cannot be attributed to overheating since the transilluminator emits negligible amounts of visible and IR radiation. Thus in contrast with the effects observed on exposure to solar radiation, the inhibition is exclusively due to UV radiation. This is important since typical photobleaching has only been studied by exposing cells to strong visible radiation [ 91. The mechanisms of UV inhibition of photo-orientation and motility are not known. However, DNA does not seem to be the primary target for UV-B
-0.600
350
400
450
500
550
Wavelength
600
650
700
i
0
[nm]
Fig. 5. Absorption spectra of a freshwater Cryptomonas species measured after increasing times of exposure to LJV radiation (from top to bottom: after 0, 145, 350, 755, 1560, 2915, 3840, 4380, 5615, 6355, 7760, 9205, 10605, 11685, 12 775, 13 960, 14 630, 16 320,17 175 and 18 990 min). llxpoaure time 50
0.
r-0
100
6 I ---o*-_o_ou-oo-~-~_~o-o-
150
1 o-
o-
[h] 200
I o-o-o-_o-oq-•-•-_0-~-0-0--
250
300
# o-o-
; -0
Q-’
>
-0.1
,s -0.2 ‘i b B a -0.3
+\+ -0.4
L+ _ ‘+ +-+-+_+
-+-+_+_
Fig. 6. Decrease in absorption at key wavelengths (representing the major photosynthetic pigments of a freshwater Cryptomonas species) due to UV bleaching after increasing times of exposure: 0,680 nm; 0,480 nm; 0,560 nm; 0,460 nm; +, 440 nm.
113
inhibition in some micro-organisms since first effects can be seen after exposure times of only a few minutes [ 10,121.In addition, photodynamic effects can be eliminated as mechanisms of inhibition by UV radiation since quenchers and scavengers of free radicals and singlet oxygen are not able to remove the inhibition in cyanobacteria and in some green and colourless flagellates [lo, 12,141. Therefore it has been speculated that an intrinsic component of the photoreceptor array and the propulsive machinery of the cell may be the targets of UV radiation causing inhibition of photo-orientation and motility respectively. The sequence of events during bleaching by UV radiation clearly demonstrates that the accessory photosynthetic pigments (phycobilins) are destroyed first, followed by the carotenoids which are believed to play a major role in photoprotection; the chlorophylls essential for photosynthesis are most resistant to UV radiation.
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (SFB 305). The authors gratefully acknowledge the skilful assistance of H. Kropfhauser, R. Krzyzanowsky, J. Schafer, K. Schmidt, D. Schnappinger, S. Seeler and B. Vollertsen. References 1 W. Nultsch and D.-P. HZder, Photomovement in motile microorganisms II, Photothem. Photobiol., 47 (1988) 837 - 869. 2 D.-P. Hiider, Ecological consequences of photomovement in microorganisms, J. Photochem. Photobiol. B, 1 (1988) 385 - 414. 3 R. Hirschberg and W. Hutchinson, Effect of chlorpromazine on phototactic behavior in Chlamydomonas, Can. J. Microbial., 26 (1980) 265 - 267. 4 D.-P. Hider, G. Colombetti, F. Lenci and M. Quaglia, Phototaxis in the flagellates, Euglena gracilis and Ochromonas danica, Arch. Microbial., 130 (1981) 78 - 82. 5 T. Ristori, C. Ascoli, R. Banchetti, P. Parrini and D. Petracchi, Localization of photoreceptor and active membrane in the green alga Haematococcus pluvialis, in Progress in Z’rotozoology, Abstracts, 6th Znt. Congress of Protozoology, Warsaw, Poland, July 5 - 11, 1981, p. 314. 6 R. Wayne, S. Roux and G. Thompson, Photomovement in Dunaliella, in M. Furuya (ed.), Phytochrome and Photomorphogenesis, Proc. 16th Yamada Conf., Okazaki, Japan, October 13 - 17, 1986, p. 103. 7 D.-P. Hader, E. Rhiel and W. Wehrmeyer, Ecological consequences of photomovement and photobleaching in the marine flagellate Cryptomonas maculata, FEMS Microbial. Ecol., 53 (1988) 9 - 18. 8 N. Ekelund and D.-P. Hader, Photomovement and photobleaching in two Gyrodinium species, Plant Cell Physiol., 29 (1988) 1109 - 1114. 9 W. Nultsch and G. Agel, Fluence rate and wavelength dependence of photobleaching in the cyanobacterium Anabaena variabilis, Arch. Microbial., 144 (1986) 268 - 271. 10 D.-P. Hiider, M. Watanabe and M. Furuya, Inhibition of motility in the cyanobacterium, Phormidium uncinatum, by solar and monochromatic UV irradiation, Plant Cell Physiol., 27 (1986) 887 - 894.
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