Photo-orientation in a freshwater Cryptomonas species

Journal of Photochemistry and Photo biology, B : Biology, 2 (1988) 123 -132

123

CRYPTOMONAS

SPECIES

PHOTO-ORIENTATION IN A FRESHWATER

ERHARD RHIEL, DONAT-P . HADERt and WERNER WEHRMEYER Instztut fiurBotamk, Staudtstr. 5, D-8520 Erlangen (F.R.G .)

(Received November 10, 1987, accepted February 23, 1988)

Keywords . Cryptomonas,

Cryptophyceae, diaphototaxis, free radicals, photodynamic responses, photomovement, phototaxis, singlet oxygen, videomicroscopy .

Summary Phototactic orientation is characterized in a freshwater species of the flagellate Cryptomonas . In contrast to the marine species, C. maculata, which shows both pronounced positive and negative phototaxis, and an unidentified red tide Cryptomonas species, which is restricted to positive phototaxis, this limnetic species possesses an unusual phototactic response : at all effective fluence rates it orients perpendicular to the direction of the light beam (diaphototaxis) . When grown under nitrogen and phosphorus deficiency, the absorption spectrum does not change considerably but the degree of orientation increases . Addition of p-quinone, a quencher of free radical formation, at concentrations of up to 10 - s M does not alter the phototactic behaviour at either high or low fluence rates, while 1,4diazobicyclo[2 .2 .2]octane (DABCO) as well as other scavengers of singlet oxygen, affects photo-orientation at high fluence rates using a concentration of 10 -4 M, but not at low fluence rates . These results indicate that the high fluence rate response may be mediated by a type II ('02 production) photodynamic effect .

1 . Introduction like many other photosynthetic and non-photosynthetic motile microorganisms, Cryptomonas has been found to respond to light in addition to other environmentally relevant stimuli [1] . A Cryptomonas species forming red tides has been shown to be restricted to positive phototaxis even at high fluence rates [2, 3] . In contrast, the marine C. maculata shows a weak positive phototaxis at fluence rates C 15 W m -2 and a more pronounced negative phototaxis at higher fluence rates [4] . The ecological importance of these two antagonistic responses is supported by the finding that in nitrogen-

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124

deficient cells, which lack part of their photosynthetic machinery [5, 6], the transition point between positive and negative phototaxis is shifted towards lower fluence rates by a factor of 5 as compared with normally pigmented cells . Neither phobic responses nor photokinetic effects have yet been found in these organisms . The action spectrum described for a red tide Cryptomonas species extends from 400 to 680 nm with a major peak at about 560 nm which corresponds to the absorption maximum of the main accessory pigment, phycoerythrin [3, 7] . The photosynthetic machinery, however, does not seem to be involved in phototactic orientation of Cryptomonas since the chlorophylls are not active in photoperception and the inhibitors of the photosynthetic electron transport chain, 3-(3',4'-dichlorophenyl)-1,1dnnethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), are not effective [3, 4] . The mechanism of light direction detection seems to be based on a periodic shading or irradiation mechanism, as described for several other flagellates which rotate during forward locomotion, such as Euglena [8, 9] and Chlamydomonas [10] . Pulsed actinic light has been found to be as effective as continuous light provided the dark interval is shorter than half the rotation time [111 . When the rotation time is increased by transferring the cells into a high viscosity medium, the critical dark interval increases accordingly [12] . The sensory transduction chain in the red tide species seems to depend on the availability of Cal' ions [12, 13] which is also characteristic for the prokaryotic gliding cyanobacterium Phormidium uncinaturn [14, 15] and the flagellate Chlamydomonas reinhardtii [16, 17] . In most cases photo-orientation is assumed to be an effective mechanism to seek sufficient fluence rates or to escape detrimental high fluence rates [18, 19] . Cryptomonads are known to be extremely sensitive to bright light exposure and to move to lower levels in the water column [20, 21] . In vitro studies have confirmed these results since exposure to continuous white light bleached and eventually killed the cells even at fluence rates as low as 5 klx [22], which is less than 10% of the fluence rate measured at the surface of a body of water at full sunlight in moderate latitudes [23] . The purpose of this study is to characterize the phototactic orientation m this freshwater Cryptomonas species [24 - 26] and to compare it with red tide and marine Cryptornonas species. Furthermore, the effects of quenchers and scavengers of free radicals and singlet oxygen are investigated in order to reveal the possible involvement of photodynamic effects in photoperception . Photodynamic effects have been found to control photo-orientation of the cyanobacterium Anabaena variabilis [27, 28] . 2. Materials and methods

2.1. Organism and culture condattons The Cryptomonas species was isolated from a freshwater pond near Marburg (Stausebach) by one of us (E .R .) and used for all experiments ; the

125 organism is currently kept in culture and can be obtained from W .W . upon request . The cells were grown at 15 °C in a Moor-Chu medium enriched with added vitamins [29] at a light/dark cycle of 14(10 h at a fluence rate of 1 .28 W mom . Preliminary experiments were performed with the cells in their original medium after 2 weeks of growth . Since N and P deficiency improved the precision of phototactic orientation, all subsequent experiments were carried out with cells transferred from the original growth medium to a Chu medium in which the Moor soil extract and the N and P sources were omitted . Cultures were grown for another 3 - 4 days before the experiments .

2.2. Measurement of photo-orientation Phototactic orientation was measured using a microcomputercontrolled image analysis system capable of automatic tracking of motile flagellates [30] . A glass cuvette with 75 mm X 8 mm X 0 .2 mm inner dimensions was filled with the culture suspension and placed on the stage of a light microscope (Zeiss Universal, Oberkochen, F .R .G .) using dark field observation in order to enhance the optical contrast . The cells were observed in an IR monitoring beam from the built-in light source in combination with an IR passing cut-off filter (RG 780, Schott & Gen ., Mainz, F.R.G .) . The movements of the cells were recorded with a conventional b/w video camera with an IR-sensitive Newvicon tube (National WV 1350 E) capable of electronic inversion of the video signal . A real time digitizer was employed to analyse the image with a spatial resolution of 512 X 256 pixels at 64 possible grey levels . The digitized image was stored in a dedicated video memory . Both the online video image and the digitized image could be observed simultaneously on separate monochrome monitors. A microcomputer (with two 8 inch flexible disk drives and a 26 MB 8 inch hard disk) had access to the video RAM in order to analyse the image. The program was designed to select an organism at random, to determine its outline and centroid (centre of gravity) and to repeat this process on the same cell until a movement vector could be calculated and stored in a disk file for further statistical treatment [31, 32] . The actinic light beam for phototactic orientation was derived from a 250 W halogen projector (Prado, Leitz, Wetzlar, F .R .G .) in combination with a heat-absorbing filter (KG 2, Schott & Gen .) . The fluence rate was controlled using neutral density absorbing type filters (Schott & Gen .) .

2 .3 . Chemicals 1,4diazobicyclo[2 .2 .2]octane (DABCO) and p-quinone were obtained from Wako (Osaka, Japan) . The first of the two quenchers is water soluble and was used by diluting a stock solution of 10 -2 M with the culture suspension to 10 -4 M . The latter was dissolved in a small aliquot of ethanol and then added to the culture medium to the final desired concentration of 5 10_ M .



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2.4. Measurement of absorption spectra The in vivo absorption spectra were determined with a scanning spectrophotometer (Hitachi U 3200) equipped with an end-on multiplier at low absorbance values to reduce spectral distortions due to scattering . The spectra were measured using cultures with about 5 X 10 5 cells ml-1-

3 . Results Preliminary experiments had shown that the degree of phototactic orientation increases in this freshwater Cryptomonas species under N and P deficiency. In order to establish that the pigment composition does not change as a consequence of the changed culture conditions, absorption spectra were measured before and after P and N deficiency (Fig . 1) . Even 4 days after transfer into the new conditions the relative pigment composition had not changed significantly, and the cell number had even increased as indicated by the overall absorption increase and cell density determination . Since the preliminary phototaxis experiments had shown a rather unusual pattern of movement perpendicular to the light direction the reliability of the tracking system needed to be proven for this organism . The best test is tracking the organisms in the absence of a light stimulus, the response to

400

500

600

700

Wave" gth lnml

Fig . 1 . In vivo absorption spectra of the freshwater Cryptomonas spec . before (lower curve) and 4 days after (upper curve) transfer into N and P deficient medium . Abscissa, wavelength (nm), ordinate, absorption .

127 which could override any bias . The histogram of movement directions tracked in darkness shows a random distribution (Fig . 2) . At all fluence rates above a threshold of about 0 .1 W m -1 the histogram was found to be bimodal with the two lobes pointing perpendicular to the light source (0 ° ) both at low fluence rates, such as 5 W m-1 (Fig . 3(a)), and at higher fluence rates (e .g . 100 W M-2 , Fig . 3(b)) . In order to study the mechanism of light direction detection, quenchers of both free radicals and singlet oxygen ('0 2 ) were employed, which are produced in type I and type II photodynamic reactions respectively . Addition of p-quinone, a scavenger of free radicals, at a final concentration of 10 -5 M did not alter the phototactic orientation of the cells at a fluence rate of 5 W m`2 (Fig . 4(a)) and the behaviour at 100 W m -2 was also hardly impaired (Fig. 4(b)) . At higher concentrations, p-quinone turned out to be cytotoxic and at lower concentrations the result did not change . DABCO, a 1 0 2 quencher, at a concentration of 10-4 M, also had no effect at 5 W m2 (Fig . 5(a)) . At 100 W in -', however, the behaviour changed drastically (Fig . 5(b)) : the perpendicular orientation was almost lost and the cells moved almost at random . Since DABCO has also been found to react with the triplet state of photosensitizers, other singlet oxygen quenchers, such as imidazole, were also studied and these showed similar effects (data not shown) . The results obtained with quenchers represent true effects of the drugs on the orientation since the cells were not noticeably affected in their swimming behaviour or velocity . 270°

180°

90 , . in the absence of a light stimulus . The Fig. 2 . Circular histogram of Cryptomonas spec total number of tracks in the histogram is 951 ; for comparison, the number of tracks in the 5 .6 ° sector centred around 90° is 15 .



128 270°

270°

so° (a)

(b)

Fig. 3. Circular histograms of Cryptomonas spec. when irradiated with (a) 5 W M-2 (the total number of tracks in the histogram is 621, the number of tracks in the 5 .6° sector centred around 90 ° is 28) and (b) 100 W M-2 from 0° (the total number of tracks in the histogram is 383, the number of tracks in the 5 .6° sector centred around 90° is 12) . 270° 270°

0

(a)

9°

(b)

s0°

Fig . 4 . Circular histograms of Cryptomonas spec . when irradiated with (a) 5 W M -2 (the total number of tracks in the histogram is 504, the number of tracks in the 5 .6 ° sector centred around 90° is 38) and (b) 100 W m -2 from 0° (the total number of tracks in the histogram is 523, the number of tracks in the 5 .6° sector centred around 90° is 16) in the presence of 10 -5 M p-quinone.

129

(a) 270'

(b)

go-

Fig . 5. Circular histograms of Cryptomonas spec. when irradiated with (a) 5 W r n-2 (the total number of tracks in the histogram is 503, the number of tracks in the 5 .6° sector centred around 90' is 21) and (b) 100 W m 2 from 0° (the total number of tracks in the histogram is 718 ; the number of tracks in the 5 .6° sector centred around 90 ° is 19) in the presence of 10 -4 M DABCO.

130 4. Discussion

In contrast to many other motile flagellates, the freshwater Cryptomoms species employed in this study showed a novel and unusual phototactic behaviour . While a red tide Cryptomonas has been reported to move exclusively positive phototactically [2] and the marine C . maculata showed both positive and negative phototaxis [3], this freshwater species oriented perpendicularly. Such behaviour has been found in a cyanobacterium at intermediate fluence rates between those causing positive and negative phototaxis [33] . It is interesting to note that various Cryptomonas species utilize such different strategies to orient in their photoenvironment . The exclusive positive phototaxis found in the red tide species will bring the population to the surface. There it is exposed to the bright sunlight fluence rates . While there are no photobleaching data for this particular species, C . maculata has been found to be extremely sensitive to prolonged exposure at even low fluence rates [22] . C . maculata escapes this hazardous situation by using an antagonism between positive phototaxis at low fluence rates and negative phototaxis at higher rates [4] which results in an accumulation at a level near the cross-over point between the two responses . A similar mechanism is used by the green flagellate Euglena, which uses negative phototaxis to move away from the water surface if the fluence rate is too bright and negative gravitaxis to move upwards [23] . The perpendicular movement with respect to the incident light beam of this Cryptomonas will keep to population at a certain depth. In the cyanobacterium Anabaena uariabilis, the direction of phototactic orientation has been found to be controlled by the fluence-rate-dependent production of '0 2 [27, 281 . 102 is produced by a photodynamic reaction with the photosynthetic pigments acting as sensitizers . Since the action spectrum of Cryptomonas phototaxis resembles the absorption spectrum of phycobilins [2] . which are also responsible for phototaxis in Anabaena, the possible involvement of type I or type II photodynamic responses [34] was studied . Since the quencher of free radicals (type I reaction),p-quinone [35, 36], is effective neither at low or high fluence rates, while 102 quenchers such as DABCO [37] disturb the high fluence rate response, the latter might be mediated by a type XI photodynamic reaction .

Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Ha 985/5-6 .-P . .H to D and SFB 305 to W.W.) and the Bundesminister ft'ir .-P The D . Forschung and Technologie under contract No . KBF 57 to .H authors gratefully acknowledge the skillful technical assistance of F . Badbanchi, K . DSrr, M . Hermans, E . Reinecke and M . Rudyk .

131

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