Photochemistry and Photobiology. 197 1 . Vol. 14, pp. 705-7 12. Pergamon Press. Printed in Great Britain

PHOTOTACTIC AND PHOTOKINETIC ACTION SPECTRA OF THE DIATOM NITZSCHIA COMMUNIS

W. NULTSCH Department of Botany, University of Marburg/L., Germany

(Received 15 January 197 1 ; accepted 3 May 197 1)

Abstract - Three different types of photomotion reactions, photokinesis, photo-topotaxis and photo-phobotaxis, have been investigated in the diatom Nitzschia communis. In photokinesis the active light appears to be absorbed by the photosynthetic pigments. Apparently, the acceleration of movement by light is due to an additional ATP supply from photosynthetic phosphorylation, as it is the case in the blue-green algae of the genus Phormidium. On the other hand, no correlations exist between photo-phobotaxis and photosynthesis, because red light does not induce photo-phobotactic responses in N. communis. The phobotactic action spectrum resembles that one of photo-topotaxis. In the visible region only violet, blue and blue-green light is active in both the reactions. However, it is not yet clear whether or not the stimuli are mediated by the same photoreceptor, mainly because of the different sensitivity of both the reactions to U.V.

INTRODUCTION

IN DIATOMS, photokinesis and phototaxis have not been investigated extensively. Heidingsfeld[l] found the following types of photic reactions in an early study of Navicula radiosa: positive and negative photo-topotaxis, i.e. a light orientated move- ment towards or away from the light source, and positive and negative photo-phobo- taxis, i.e. a light induced reversal of the direction of movement caused by a sudden decrease or increase in light intensity. In photo-topotaxis she found blue and violet light (A < 480 nm) were active, while she observed photo-phobotactic reactions in blue and red light as well.

More detailed investigations were carried out by Nultsch [2] in several species of the genera Navicula, Nitzschia and Amphora. Using interference filters, he observed only positive photo-topotactic reactions at wavelengths between 400 and 550 nm. The same range of the visible spectrum was active in photo-phobotaxis, while in light be- tween 550 and 720 nm accumulation as well as migration out of the light field occured. Furthermore, he observed an effect of light on the speed of movement, called photo- kinesis. These findings pointed to a possible role of yellow pigments in photo-topo- taxis, while in photo-phobotaxis correlations with photosynthesis could not be ex- cluded. In order to obtain more information, we measured the action spectra of both photo-topotaxis and photo-phobotaxis in the diatom Nitzschia communis. We observed a significant effect of light on the speed of movement in the sense of positive photo- kinesis, i.e., an acceleration of movement by light. Because photokinesis in diatoms has not been studied so far, we also investigated the photokinetic phenomena in Nitzschia communis.

MATERIALS AND METHODS

The diatom Nitzschia communis Rabh. was isolated from soil of the Botanical Garden in Tuebingen. I t has been kept in axenic cultures since 1963 in the following

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medium:

0-2 g K,HPO,, 0.1 g Ca(NO,), X 4 HzO, 0.05 g MgSO, X 7 H 2 0 , 0.6 ml Si(OH),, 6.95 mg FeSO, X 7 H,O, 152 pg H,BO,, 287 ~ . lg ZnSO, X 7 H,O, 169 p g MnSO, x H,O, 2.49 pg CuSO, X 5 H,O, 12.35 pg (NH,)6Mo,0,, X 4 H,O, 16 pg KI, 12 p g KBI-, 21 pg Sr(N03),, 28 p g CoSO, X 7 H 2 0 , 24 pg AICl, x 6 H,O, 12 pg RbCl, 1000 ml distilled water.

The cells of N. communis are 17-23 pm long and 3 to 4.5 pm wide. Auxospore formation has not been observed. N. communis was cultivated at constant temperature (25°C) and at light intensities of 1500 lux.

For irradiation, an interference filter monochromator system has been used, as described by Mohr and Schoser[3]. The approximate half hand widths of the inter- ference filters used was 10 nm.

RESULTS

Photokinesis. The most frequently used method to evaluate the photokinetic effect in microorganisms is to measure the velocity of single cells microscopically under diff- erent light conditions. However, in slowly moving organisms, such as diatoms and blue- green algae, it is more convenient to measure the speed of a population spreading over a microscopic slide or an agar plate[4]. Initially, both the methods were used. Since they gave the same results, the second method was preferred.

The speed of movement of N. communis, which is motile in the dark, is increased by white light. The maximum of acceleration or, in other words, the photokinetic optimum [ 5 ] , is reached at about 500 lux. With further increasing light intensity the acceleration of movement diminishes again. At 500 lux the speed is twice as in the dark.

The action spectrum of photokinesis (Fig. l ) , measured at 28 X lo-" Einstein cm-2 sec-', displays a strong effectiveness of red light, with maximum activity at about 670 nm. The shoulders at 620 and 700 nm may not be significant. The range of photokinetic activity extends to 800 nm. This surprising result is consistent with former observations in blue-green algae of the genus Phormidium, in which far red light was also photo- kinetically active [4].

Since the maximum at 670 nm is probably due to chlorophyll a , the action of blue light with a maximum between 430 and 440 nm may be ascribed to the Soret band of chlorophyll a. However, the height of the blue band is much less than would be ex- pected from absorption spectrum of chlorophyll a.

Contrary to the blue-green algae[6], U.V. is slightly effective. The minimum in the action spectrum between 480 and 500 nm indicates that carotenoids in general are not involved in absorption of the photokinetically active light. However, the effectiveness of light between 500 and 550nm suggests a possible role of fucoxanthin as a photo- receptor of photokinesis, which the slight shoulder at 540 nm in the in v i m absorption spectrum (Fig. 1) is due to.

Photo-topotaxis. If diatoms or blue-green algae are transferred to a slide or an agar plate, they spread circularly from the inoculation spot under diffuse light. However, under unilateral illumination the spreading area is shifted toward the light source in case of positive photo-topotaxis, forming an ellipse. The extent of shifting, corrected

Phototactic and photokinetic action spectra

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Fig. 1. Photokinetic action spectrum (solid 1ine)'and in uiuo absorption spectrum (dotted line) of N . communis. Abscissa: wavelengths; ordinate left: photokinetic response in relative units;

right: absorbance.

to eliminate a possible distortion by photokinesis [7], has been used as an index of the photo-topotactic response.

In white light, N. communis displays only positive topotactic reactions up to 40,000 lux, with an optimum at 200 lux (Fig. 2). The zero threshold lies at 2 lux. The action spectrum of photo-topotaxis was measured at 25 X lo-" Einstein cm-2sec-1. As shown in Fig. 3, N. communis displays positive reactions over the whole spectral range be-

40

30

20

10

. , 1 2 5 1 0 2 I lux

Fig. 2. Photo-topotactic reaction in white light. Abscissa: light intensity; ordinate: topotactic response in relative units.

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tween 33 5 and 550 nm. The three peaks at 380,430 and 490 nm may not be significant. Beyond 500 nm the photo-topotactic activity decreases markedly, and wavelengths longer than 550 nm are quite ineffective. These findings are consistent with results of our former semiquantitative experiments on the spectral photo-topotactic sensitivity of several diatoms [3].

The strong activity of violet, blue and blue-green light up to 550 nm suggests the role of carotenoids as photoreceptors in photo-topotaxis of diatoms. However, caro- tenoids would not account for the strong topotactic effect of the near U.V. Thus, the problem seems to be similar to that in photo-topotaxis of flagellates or in phototropism.

Photo-phobotuxis. For the evaluation of photo-phobotactic reactions the ‘light trap’ method of Engelmann[8] was used. In this method a light field is projected by a slit onto a homogeneous preparation of diatoms on an agar plate or a microscopic slide[3]. In case of positive reaction the cells accumulate in the light trap (Fig. 4a).

Since diatoms move slowly, the number of cells in the light trap increases during the first hours of the experiment until a constant level is reached after 6-8 hr (Fig. 5 ) which represents the maximum number of organisms that can collect in the light field under the given light conditions. This level is evaluated by measuring the optical density of the accumulated organisms at the end of the experiment, after removing the slit [5. 91.

The photo-phobotactic sensitivity is decreased by preillumination and increased by dark adaptation of the organisms. As shown in Fig. 6, the number of reacting organisms increases with increasing time of dark adaptation to a certain extent, which depends on the light conditions before. Maximum sensitivity is reached after about 8 hr. There- fore, in all photo-phobotactic experiments the diatoms were kept in the dark for 8 hr before the experiments.

Fig. 3. Photo-topotactic action spectrum of N . communis. Abscissa: wavelength; ordinate: topotactic response in relative units.

: , . r f

, - . *

(a 1 (b)

Fig. 4. Photographs of the light field after removing the slit. (a) Accumulation of diatoms in the light trap as a result of positive photo-phobotaxis. (b) Combination of positive photo-phobo- tactic reaction, and positive photo-topotaxis. (c) Migration out of the light field after irradia-

tion with red light (670 nm).

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Phototactic and photokinetic action spectra

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80-

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Fig. 5 . Increasing density of organisms in the light trap with time. Abscissa: time of irradiation; ordinate: density of light field in relative units. Light intensity: 500 lux.

l0L------ 0 1 2 3 L 5 6 7 8 9 10h

Fig. 6. Effect of dark adaptation on the strength of photo-phobotactic reactions. Abscissa: duration of dark adaptation: ordinate: density of light field in relative units after irradiation

(8 hr) with 500 lux.

In white light, the threshold of photo-phobotaxis was found at 2 lux (Fig. 7). With increasing light intensities the density of the accumulated organisms increases up to 1000 lux. Above 1000 lux, the light trap method fails to give correct results, because of photo-topotactic effects. The diatoms in the vicinity of the light field are stimulated photo-topotactically by laterally scattered light [5 ] to enter the light field or to form a more or less broad ring zone surrounding the light field (Fig. 4b).

Therefore, to avoid any superimposition of phobotaxis by topotaxis, the action spectrum was measured at very low intensities, about 1 O-" Einstein cm-2sec-1. While in violet and blue light typical positive photo-phobotactic reactions lead to an accumula- tion of organisms in the light field (Fig. 4a), in red light the organisms leave the light field (Fig. 4c). The migration out of the light field, however, cannot be considered to

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Fig. 7. Photo-phobotactic reaction in white light. Abscissa: light intensity: ordinate: photo- phobotactic reaction in relative units.

result from negative photo-phobotaxis, because negative responses, i.e., reversals induced by an increase in light intensity, in red light have never been observed micro- scopically.

As shown in Fig. 8, the whole range of wavelengths between 380 and 550nm is active. We find a broad band of maximum activity between 420 and 490 nm, without significant peaks at any wavelength. Beyond 490 nrn, the photo-phobotactic activity decreases, and wavelengths longer than 560 nm are quite ineffective.

Although the number of individuals which left the light field when longer wave- lengths were used was not measured, a rough estimation revealed that the migration out of the light trap was maximal at 670 nm and followed an action spectrum similar to that of photokinesis.

400 450 500 550 nm

Fig. 8. Photo-phototactic action spectrum of N . communis. Abscissa: wavelength: ordinate: photo-phobotactic response in relative units.

Phototactic and photokinetic action spectra 71 1

Since the migration out of the field, observed in red light, cannot be considered to result from photo-phobotactic reactions, chlorophyll a is not a photoreceptor of photo- phobotaxis. Consequently, there are no correlations between photo-phobotaxis and photosynthesis in diatoms. On the contrary, in diatoms the action spectra of photo- topotaxis and photo-phobotaxis are rather similar, with the exception of the near U.V. Therefore, it seems to be possible that both the reactions are mediated by the same photoreceptor in the visible region, a situation, which resembles that one in flagellates. However, whether carotenoids or flavoproteins are the photoreceptors cannot be con- cluded from these action spectra.

DISCUSSION While photokinesis in N. communis, as in blue-green algae and purple bacteria,

appears to be due to an additional supply of ATP from photosynthetic phosphorylation, correlations between photo-phobotaxis and photosynthesis can be excluded because of the phobotactic ineffectiveness of red light. This is quite different from the situation in purple bacteria and blue-green algae.

Obviously two types of photo-phobotactic reaction mechanisms exist in micro- organisms:

(1) The bacterial type, which is realized in prokaryonta, i.e., in phototrophic bac- teria and in blue-green algae. In this type, photo-phobotaxis is coupled with photo- synthesis [ 10, 1 11, and the photo-phobotactic responses are obviously due to transient changes in the photosynthetic electron flow caused by sudden changes in light intensity.

(2) The flagellate type, which is realized in eukaryontic cells, e.g. in flagellates and, according to the forementioned results, in diatoms. In this type only light of wave- lengths shorter than 550 nm is active. Consequently, the photo-phobotactic stimulus cannot be mediated by the photosynthetic apparatus.

In the second type, the action spectra of photo-phobotaxis and photo-topotaxis are similar. For this reason these two reaction types have been confused very often with each other. However, whether or not the photoreceptors of both the reactions are identical, cannot be concluded from these action spectra, especially because of diff- erences in the effectiveness of the near U.V.

The chemical nature of the photoreceptor is not yet known. Because of the strong activity of violet and blue light, carotenoids and flavoproteins must be considered. On the one hand, the effectiveness of radiation between 3 50 and 400 nm, which is scarcely absorbed by carotenoids, is an argument for the photoreceptor role of flavoproteins, although under certain circumstances even carotenoids display absorptions peaks be- tween 360 and 380 nm[12]. On the other hand, the activity of light between 500 and 550 nm, which is poorly absorbed by most flavoproteins but is absorbed by fucoxanthin, favours the possible role of carotenoids in the perception of the phototactic stimulus. Thus, this important question remains undecided now as before. Further investigations, especially in flagellates, will be necessary.

A possible explanation for the observation that in bacteria and blue-green algae the photosynthetic apparatus is active in mediating the photo-phobotactic stimulus, while in eukaryonta no correlations at all exist between photo-phobotaxis and photo- synthesis, may be the existence of a chloroplast envelope. This envelope separates the photosynthetic apparatus from the cytoplasm in eukaryonta and may partially isolate

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the other cell compartments from the effects of photosynthesis. In prokaryonta the thylakoids are located in the cytoplasm, and any change in the steady state of the photo- synthetic electron flow may be transmitted to the surrounding cytoplasm and the cyto- plasmic membrane, causing a change in the membrane potential, as it has been suggested by Nultsch [ 5 ] .

Acknowledgements -This investigation has been supported by the Deutsche Forschungsgemeinschaft. I am indebted to Miss W. Hauber for valuable technical assistance.

REFERENCES 1. 1. Heidingsfeld, Thesis, Breslau (1943). 2. W. Nultsch,Archio Proristk. 101, 1 (1956). 3. H. Mohr and G. Schoser, Plunra (Berl.) 53,l (1959). 4. W. Nultsch,Plunta 57,613 (1962). 5. W. Nultsch, In Photobiology ofMicroorgnnisms, (Edited by P. Halldal) Wiley, New York ( 1 970). 6. W. Nultsch,Ber. dt. bot. Ges. 75,443 (1962). 7. W. Nultsch, Plunta 56,632 (1961). 8. Th. W. Engelmann, PJiigersArch. ges. Physiol. 29,387 (1882). 9. W. Nultsch, Plunta 58,647 (1962).

10. R. K. Clayton, In Handbuch der PJunzenphysiologie (Edited by W. Ruhland), Vol. 1711, 371, Springer

1 1 . W. Nultsch and G. Richter,Arch. Mikrobiol. 47,207 (1963). 12. A. Hager, Plunta 91,38 (1970).

Berlin, Gottingen, Heidelberg (1959).