Planta (Berl.) 92, 259--266 (1970)

The Circadian Rhythm of a Behavioral Photoresponse in the Dinoflagellate Gyrodinium dorsum* RICHARD B. FORWARD, JR.~ and DE~IO~EST DAVENPORT Department of Biological Sciences, University of California, Santa Barbara Received January 6/March 16, 1970

Summary. The circadian rhythm of the photoresponse to blue light in the dinoflagellate GyrodiniumdorsumKofoid was investigated by the use of a closed circuit television system. The initial cessation of movement upon stimulation (stop-response) was used as the index of light reception. Under constant dark conditions cells grown on a 12L: 12D regime show an endogenous circadian rhythm in their stop-response with maximum responsiveness occurring approximately one hour before the beginning of the expected light phase. This rhythmic response was only observed if the cells were irradiated with red light (620 nm) prior to stimulation with blue light. After preirradiation both far-red reversibility and the shift in the stop-response action spectrum from 470 nm to 490 nm could also be demonstrated. These findings may be related to the diurnal migration of marine dinoflagellates. Introduction Physiological phenomena which occur rhythmically have long been known to occur in phytoflagellates, e.g. in photosynthesis in Gonyaulax polyedra (Sweeney, 1960) and in bioluminescence in Gonyaulax (Hastings and Sweeney, 1958). These, together with Pohl's (1948) demonstration of a circadian r h y t h m in the phototaxis of Euglena, suggest that a similar r h y t h m might also occur in the photoresponses of dinoflagellates. The dinoflagellate Gyrodinium dorsum Kofoid responds to light stimulation by initially ceasing movement (stop-response), and then swimming in the direction of the light source (phototaxis). For both of these phases of the response, the action spectra maximum is at 470 nm (Hand etal., 1967). The stop-response has been shown to involve a phytoehrome pigment, since prior irradiation with red and far-red light alter the responsiveness to 470 nm light (Forward and Davenport, 1968). Further investigation has shown that following a red (620 nm) irradiation, the threshold for responsiveness is lowest for 470 nm light, and after a far-red (700 nm) exposure, the cells are most sensitive to 490 nm (For. ward, 1970). The initial search for a r h y t h m in this photoresponse of Gyrodinium was unsuccessful (Collard, 1967), but this study was performed before the * This study was supported by National Science Foundation grant GB 5137.

260

R.B. Forward, Jr., and D. Davenpoit:

i n t e r a c t i o n of red, f a r - r e d a n d blue ligh~ h a d been esgablished. I n t h e e x p e r i m e n t s which follow, t h e existance of a c i r c a d i a n r h y t h m is established, a n d its r e l a t i o n to r e d i l l u m i n a t i o n is also i n v e s t i g a t e d .

Materials and Methods The culturing procedure remains as reported by Hand et al. (1967). A 1.5 m 1 aliquot sample was pipetted into a well slide (Hand et al., 1967) which was placed on the stage of a Nikon inverted microscope (l~odel M). A Cohu series 3000 television camera was coupled to the side camera mount of the microscope, and the recorded picture displayed on a Cohu model CNB-8/RBL video monitor. The cells were observed in dark field illumination, the light of which was filtered with a 730 nm interference filter (wavelengths in this region did not influence the light response). A Bausch and Lomb No. 33-86-02 grating monoehromater was used as the source of light stimulation (Forward, 1970). Stimulus duration was controlled by a Compur shutter and was arbitrarily set at 1 sec. The initial cessation of movement or stop-response, used as the indicator of light reception, was recorded by photographing the video monitor. Since the response latency was 0.4---0.6 sec (Hand et al., 1967), the picture was taken by the camera 2/3 see after the beginning of light stimulation. By using a 1/4 see camera exposure, moving cells appear on film as long blurry lines and stopped cells as round or oval dots. The general experimental procedure was as follows. Cells grown on a 12 hr light: 12 hr dark (12L: 12D) regime were either retained on this schedule or placed in constant darkness, i.e. a light phase was never given. Aliquot samples were removed from cultures every few hours, and the threshold intensity of a positive stopresponse to blue light was determined. A positive stop-response is arbitrarily designated as a level of stopping above 50% (Forward and Davenport, 1968).

Data

1. Circadian Rhythm /or Response to 470-nm Light. I t w~s first necessary to d e t e r m i n e t h e responses of cells to 470 n m s t i m u l a t i o n when growing u n d e r a 1 2 L : 1 2 D regime. D u r i n g t h e n i g h t phase a positive stop-response could n o t be i n i t i a t e d w i t h a n y i n t e n s i t y of 470 n m (Fig. 1). W h e n t h e lights c a m e on, t h e t h r e s h o l d decreased r a p i d l y , a n d after a b o u t 3 h r t h e cells a t t a i n e d t h e i r g r e a t e s t sensitivity. T h e t h r e s h o l d r e m a i n e d a t this low level u n t i l t h e culture lights w e n t off, a f t e r which t i m e t h e cells were no longer responsive. The critical question was w h e t h e r t h e cells w o u l d become responsive d u r i n g t h e d a y phase if t h e lights r e m a i n e d off. Consequently, t h e ceils were left in c o n s t a n t darkness, a n d t h e p r o c e d u r e of t h e first e x p e r i m e n t r e p e a t e d . A positive stop-response was n e v e r o b s e r v e d u n d e r t h e s e conditions to a n y s t i m u l a t i o n i n t e n s i t y . Since previous w o r k h a d d e m o n s t r a t e d t h a t s e n s i t i v i t y to 470 n m was g r e a t l y i n c r e a s e d b y p r i o r e x p o s u r e to r e d light a n d decreased b y f a r - r e d l i g h t ( F o r w a r d a n d D a v e n p o r t , 1968), t h e s t i m u l a t i o n p r o c e d u r e

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Fig. i. Response to 470 nm light stimulation of eelIs grown on a I2L:12D cycle over a 24 hr period. Ordinate: threshold intensity at 470 nm necessary for im'~iating a positive stop-response. Intensity represented a~ percent of 3.07 • 10~5quanta cm-z see-~, "None" indicates no 4/0 nm light intensity could initiate ~ positive stop-response. Abscissa: time during the 24 hr period. D~rk phase w~s from 00:00 to 12:00 o'clock as shown by heavy dark line. @~ response to 470 nm. o ~ response to 470 nm after ~ 45 sec of irradiation with 620 nm (8.1 X 1015quanta cm-~ see-l). E~eh point is the average of the threshold determinations for that time (minimum of 3 iris}s) was modified so t h a t cell samples were r e m o v e d from the culture chamber every few hours during the d a y and night, illuminated for 45 see with 620 nm, a~d placed in darkness. A n energy threshold for a response to 470 n m was then determined. The reponses resemble those for blue stimulation alone except that, the cells began responding about 3 hr before the begiIming of the light period, showing increasing sensitivity ~s d a w n approached (Fig. 1). W h e n cells were kept in continuous darkness and samples stimulated as in the previous experiment, a circadian r h y t h m for the stop-response was seen (Fig. 2). During t h e first p a r t of the night period the cells were unresponsive, b u t as dawn approached t h e y became responsive, reaching their lowest threshold about 1 hr before the light period was due to begin. The threshold began to rise shortly after theoretical dawn and within 5 hr the cells ceased responding. This condition persisted until the theoretical time for the begim~ing of the second light phase when t h e y became responsive again, exhibiting m a x i m u m responsiveness a b o u t 1 hr before dawn. Therefore, a circadian r h y t h m in the stop-response of Gyrodi~ium does appear to exist, showing a peak shortly before dawn a n d having a period of about 24 hr.

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Fig. 2. Response of cells to 470 nm stimulation after illumination with 45 sec of 620nm light (8.1x101~ quanta cm-2sec-1). Cells grown on 12L-12D cycle and response tested over 48 hr in constant darkness. Normal light phase should have been from 12:00 to 24:00 o'clock. Ordinate and abscissa as described in Fig. 1

2. Circadian Rhythm o/ Responsiveness to 490-nm Light. Previous work demonstrated that following an exposure to red light which presumably converts the phytochrome into the Pfr state, the cells show greatest sensitivity to stimulation with 470 nm. After a far-red light exposure they are maximally responsive to 490 nm. If Pr is the stable form of the phytochrome to which Pfr reverts in darkness, as is the case in m a n y plants (Taylor, 1968), it might possibly be present over the 24 hr light-dark period, thereby permitting the cells to respond to 490 nm. To test this assumption the cells were placed in constant darkness and responsiveness to 490 nm tested over the day. A positive stop-response was never observed upon stimulation with any 490 nm intensity. Assuming t h a t far-red light converts the phytoehrome into the Pr form, possibly prior exposure to 700 nm is necessary for a response to 490 nm. I n continuous darkness responsiveness was tested by illuminating with 45 sec of 700 n m and then stimulating with 490 nm fight. Gyrodinium again failed to show a positive stop-response throughout the entire day. The cells, however, do show a circadian r h y t h m in their response to 470 nm after exposure to red light (Fig. 2). Also, previous work indicates t h a t red light can initiate a prolonged response to 490 nm (Forward, 1970). Therefore, exposure to 620 n m m a y be necessary for a 490 n m initiated response. Cells were grown on the 12L: 12D regime and placed in continuous darkness. At different times throughout the day samples were given a 45 sec exposure to 620 rim, after which the energy threshold

Circadian Rhythm in Gyrodinium dorsum

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Fig. 3. Response of cells to 490 nm after a 45 sec irradiation with 620 nm light (8.1 x 1015 quanta cm -~ sec-1) (.) and after a similar red exposure followed by 45 sec of 700 nm (9.8 x 1015 quanta cm -2 sec-1) (9 Cells grown on a 12L: 12D cycle and then placed in constant darkness. Ordinate: energy threshold of 490 nm necessary for initiating a positive response. Intensity represented as percent of 3.88 x 1015 quanta cm-2 sec-1. "None" indicates no 490 nm intensity could initiate a positive stop-response. Abscissa: time during the 24 hr period. Light phase would have occurred from 12:45 to 01:00. Each plot is the average of the threshold determinations for that time (minimum of 3 trials)

for a response to 490 n m was d e t e r m i n e d . The cells b e g a n r e s p o n d i n g a b o u t 3 hr before t h e o r e t i c a l dawn, r e a c h e d t h e i r lowest t h r e s h o l d level a t a b o u t d a w n a n d ceased r e s p o n d i n g w i t h i n 5 h r (Fig. 3). This response p a t t e r n e x a c t l y parallels t h a t f o u n d for s t i m u l a t i o n w i t h 470 n m (Fig. 2). T h e threshold, levels, however, were higher for t h e 490 n m response. These d a t a could result from s t i m u l a t i o n with a less effective wavel e n g t h of a n a c t i o n s p e c t r u m h a v i n g a p e a k a t 470 nm. Therefore, t h e r h y t h m was r e - e x a m i n e d for t h e effect of following t h e r e d exposure w i t h f a r - r e d fight, since 700 n m reduces s e n s i t i v i t y t o 470 n m a n d increases responsiveness t o 490 n m ( F o r w a r d , 1970). G r o w t h conditions r e m a i n e d as in t h e previous e x p e r i m e n t s a n d t h e t h r e s h o l d for a response to 490 n m was d e t e r m i n e d t h r o u g h o u t t h e d a y , after samples were i l l u m i n a t e d c o n s e c u t i v e l y b y 45 sec of 620 nm, t h e n 45 see of 700 nm. The r h y t h m i c response of t h e cells (Fig. 3) was similar to t h a t shown in t h e previous e x p e r i m e n t , b u t t h e t h r e s h o l d values are slightly lower a f t e r t h e f a r - r e d light exposure. Therefore, i t a p p e a r s t h a t r e d light i l l u m i n a t i o n is necessary for ac~ivathlg t h e p h o t o r e s p o n s e in Gyrodinium, b u t s u b s e q u e n t e x p o s u r e to f a r - r e d light can m o d i f y t h e responsiveness. T h e p e a k s in t h e c i r c a d i a n r h y t h m for a response to 470 a n d 490 n m occur a t e x a c t l y t h e s a m e time, i.e. w i t h i n t h e h o u r before dawn. U n d e r

264

R.B. Forward, Jr., and D. Davenport:

c o n s t a n t d a r k conditions, it is a s s u m e d t h a t this is t h e t i m e of g r e a t e s t responsiveness. Therefore, t h e difference in s e n s i t i v i t y b e t w e e n s t i m u l a tion w i t h 490 a n d 470 n m a t this t i m e i n t h e l i g h t - d a r k cycle following e x p o s u r e t o r e d a n d f a r - r e d light was t e s t e d . Cell s a m p l e s were i r r a d i a t e d w i t h 620 n m for 45 sec, a n d in s u b s e q u e n t d a r k n e s s a n e n e r g y t h r e s h o l d was d e t e r m i n e d for a response t o 470 nm. I m m e d i a t e l y , t h e p r o c e d u r e was r e p e a t e d , a n d a similar d e t e r m i n a t i o n m a d e for 490 a m . T h e t i m e b e t w e e n t h e t w o e x p e r i m e n t s was n e v e r longer t h a n 10 rain. Since s e n s i t i v i t y v a r i e d b e t w e e n cultures a n d w i t h t i m e d u r i n g t h e h o u r before dawn, t h r e s h o l d values can o n l y be c o m p a r e d w i t h i n each culture. Responses to 470 n m a l w a y s h a d lower e n e r g y t h r e s h o l d s t h a n those t o 490 n m (Table 1). Table 1. Threshold determin.~tion /or stimulation alter illumination with 45 see 620 n m (8.1 • 1015 quanta em -~ sec-1)

The thresholds are shown for each culture; The figures are quanta em-~see-1 • 1015 Culture

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Table 2. Threshold determination/or stimulation alter illumination with 45 sec 620 n m (8.1 • 1015 quanta em -2 aec-1) and then 45 sec 700 n m (9.8 • 10 ~ quanta cm -2 sec-1)

The thresholds are shown for each culture, in quanta cm-2sec-l• 10TM. "None" indicates no intensity of light from the monoehromator could initiate a positive stop-response. Culture

Threshold 470 nm

Threshold 490 nm

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2.34 3.07 3.07 None None

2.45 1.94 2.96 2.45 1.94

T h e e x a c t e x p e r i m e n t a l p r o c e d u r e was r e p e a t e d e x c e p t t h a t a 45 see i r r a d i a t i o n to 700 n m was i n t e r p o s e d b e t w e e n t h e r e d e x p o s u r e a n d t h e t h r e s h o l d d e t e r m i n a t i o n s . I n all cases e x c e p t one (Table 2), t h e t h r e s h o l d for a response to 490 n m was lower t h a n t h a t to 470 a m . C o m p a r i s o n of Tables 1 a n d 2 show t h a t response t h r e s h o l d s for 470 n m

Circadian Rhythm in Gyrodiniumdorsum

265

were lowest after a red light exposure, and the 490 nm response thresholds were slightly lower after a far-red exposure. Discussion

The photoresponse of Gyrodinium clearly occurs as a circadian rhythm. When grown on a 12 L: 12 D cycle the ceils showed a maximum stop-response during the day phase and no response at night. Such a response difference persisted under constant dark conditions with maximum responsiveness at the time of theoretical dawn. However, the initiation of a response under these conditions depended upon prior exposure to red light. Once irradiated with red light, far-red reversibility and the blue action spectrum shift from 470 to 490 nm could be demonstrated, but without this exposure a positive stop-response was never observed. A similar r h y t h m involving red and blue light occurs for phototaxis of the desmid Micrasterias (Nenseheler, 1967). In this ease, however, peak responsiveness occurs in the middle of the light phase, and photosynthetic pigments may be involved. In Gyrodinium the response action spectra plus reversibility by red and far-red light detract from the suggestion that chlorophylls participate directly in the photoresponse. Gyrodinium grown on a 12L:12D cycle became responsive shortly before dawn and remained so throughout the entire day phase. Under constant dark conditions, however, red light can activate responsiveness only in the 6 hr interval centering around dawn. Therefore, an intriguing question arises as to how the response is maintained in the presence of the white light in the growth chambers (Fig. 1). Collard (1967) demonstrated that Gyrodinium grown under continuous low-intensity light remains maximally responsive throughout an entire day. Also once the cells are removed from under the lights, responsiveness disappears in a few minutes (Forward and Davenport, 1968). A possible explanation is that after activation by red light, the continual shifting of the phytochrome by red and far-red light maintains the potential for a response to blue light. Support for this idea comes from the demonstration of phytochrome participation in the response (Forward and Davenport, 1968). The cells do not respond to 470 nm after a 4 min exposure to this wavelength. Subsequent irradiation with 620 nm causes the stop-response to reappear, but it can only be elicited in the first 7 min after the red exposure. If the red exposure is foliowed by far-red light, the subsequent response is shortened to 2 miu. Consecutive red and far-red exposure always shows that after a terminal red light exposure the response lasts 7 rain and after far-red 2 rain. Therefore, reversal by red and far-red light appears to maintain the response, and the terminal exposure determines the response length in succeeding darkness.

266

Forward, Jr., and Davenport: Circadian Rhythm in Gyrodinium dorsum

B o t h the phototactie m o v e m e n t t o w a r d the stimulating light which occurs after the stop-response, and the oecuranee of peak responsiveness for the stop-response at dawn suggest a relationship between the entire photoresponse and diurnal vertical migration of dinoflagellates to the ocean surface during the day. Although such a migration has not be studied in Gyrodini~m, it has been noted in other dinoflagellates (Hasle, 1950, 1954). Light oriented m o v e m e n t is u n d o u b t e d l y significant in diurnal migration, although phototaxis in response to changes in daylight intensity m a y no~ be the only process involved (Eppley et al., 1968). Nevertheless, the oceurance of the m a x i m u m for the circadian r h y t h m in phototaxis b y Gyrodinium at the beginning of the light period does correlate with a beginning of u p w a r d migration at dawn. Field studies are currently in progress to verify the existence of such a diurnal migration in Gyrodinium. R e f e r e n c e s

Collard, P.A.: An investigation of rhythmicity in the stop response of Gyrodiniu~n dosur~ Kofoid. Master's Thesis. University of California, Santa Barbara (1967). Eppley, R.W., Holm Hansen, 0., Strickland, J. D. H. : Some observations on the vertical migration of dinoflagellates. J. Phycol. 4, 333--340 (1968). Forward, R.B., Jr.: Change in the photoresponse action spectrum of the dineflagellate Gyrodinium dorsu~nby red and far-red light. Planta (Berl.) 92 HS 997 (1970). - Davenport, D. : Red and far-red light effects on a short-term behavioral response of a dinoflagellate. Science 161, 1028--1029 (1968). Hand, W. G., Collard, P.A., Davenport, D. : The effects of temperature and salinity change on swimming rate in the dinoflagellates Gonyaulax and Gyrodinium. Biol. Bull. 128, 90--101 (1965). --Forward, R.B., Davenport, D.: Short-term photic regulation of a receptor mechanism in a dinoflagellate. Biol. Bull. 138, 150--165 (1967). tIasle, G.R.: Phototactic vertical migration in marine dinoflagellates. Oikos 2, 162--175 (1950). - More on phototactic diurnal migration in marine dinoflagellates. Nytt Magsin Bet. 2, 139--147 (1954). Hastings, J., Sweeney, B. : A persistent diurnal rhythm of luminescence in Gonyaula~ 19olyedra. Biol. Bull. 115, 440--458 (1958). Neuscheler, W.- Bewegung und Orientierung bei Micrasterias dentrivulata Breb. im Licht. II. Photokinesis und Phototaxis. Z. Pflanzenphysiol. 57, 151--t72 (1967). Pohl, R.Z.: Tagesrhythmus im phototaktischen VerhaRen der Euglena gracilis. Z. Naturforsch. 3b, 367--374 (1948). Sweeney, B.M. : The photosynthetic rhythm in single cells of Gonyaulaxl~otyedra. Cold Spr. Harb. Syrup. quant. Biol. 25, 145--148 (1960). Taylor, A.O.: In vitro phytochrome dark reversion process. Plant Physiol. 48, 767--774 (1968). Richard B. Forward, Jr. Department of Biology Kline Biology Tower Yale University New Haven, Connecticut 06520, USA