Stimulable and Spontaneous
Bioluminescence in the Marine
Dinoflagellates, Pyrodinium
bahamense, Gonyaulaxpolyedra,
and Pyrocystis lunula
W. H. BI G G L E Y , E. S W I F T , R . J . B U C H A N A N , and H. H. S E L I G E R
From the McCoUum-Pratt Institute and the Department of Biology,and the Chesapeake Bay
Institute, The Johns Hopkins University, Baltimore, Maryland 21218. Dr. Swift's present
address is Graduate School of Oceanography, University of Rhode Island, King~ton, Rhode
Island 02881. Dr. Buchanan's present address is Department of Civil Engineering, University of Washington, Seattle, Washington 98105
ABSTRACT P. bahamense, G. polyedra, and P. lunula exhibit interspecies differences in stimulable and spontaneous bioluminescence. For each species the total
number of photons that can be emitted upon mechanical stimulation is a constant, regardless of the time during scotophase at which stimulation occurs.
Ratios of stimulable bioluminescence per organism during scotophase and photophase are as high as 950:1 for laboratory cultures and have been observed as
high as 4000: 1 for natural populations of P. bahamense. Spontaneous emission in
darkness shows flashing as well as low-level continuous emission. Natural populations of P. bahamense, placed in darkness during natural photophase, exhibit a
dual character to their stimulable bioluminescence. Mechanical stimulation
techniques are described for rapid and reproducible stimulation of bioluminescence.
INTRODUCTION
I n previous papers dealing w i t h the n a t u r a l r h y t h m s of bioluminescence of
the tropical m a r i n e dinoflagellate Pyrodinium bahamense (Seliger et al., 1962;
T a y l o r et al., 1966; Seliger a n d McElroy, 1968), b o t h the shapes of the daily
stimulable bioluminescence curves a n d the night-to-day ratios of stimulable
bioluminescence differed m a r k e d l y from the results reported by Hastings
a n d Sweeney (1957, 1958, 1959), Sweeney a n d Hastings (1957, 1958, 1960),
a n d Sweeney et al. (1959) for laboratory cultures of a different species,
Gonyaulax polyedra.
96
The Journal of General Physiology
W.
H.
BIOGLEY
ET
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Stimulable and Spontaneous Bioluminescenee
97
We therefore established unialgal laboratory cultures of P. bahamense and
G. polyedra, as well as a third nonmotile species, Pyrocystis lunula, and developed
precise mechanical stimulation and light measurement techniques. We have
made a comparative study of the bioluminescence of these three species under
identical conditions of laboratory culture as well as of the bioluminescence of
natural populations of P. bahamense. The present paper describes our techniques and the results of these comparisons.
M 'i A T E R I A L S
AND
METHODS
A. Dinoflagellate Cultures
Table I lists the data relative to the origins and culture conditions for the three species of dinoflagellates investigated. Only actively growing, log-phase cultures were
TABLE
I
ORIGINS AND CULTURE CONDITIONS FOR DINOFLAGELLATES
Organism and habitat
P. bahamense Plate, armored
dinoflagellate, shallow, welllit bays of West Indies
G. polyedra Stein, armored dinoflagellate, cosmopolitan
P. lunula Schiitt, unarmored
dinoflagellate, cosmopolitan
Isolation
B.M. Sweeney, 1966, Oyster
Bay,
Jamaica,
West
Indies
B.M. Sweeney, 1952,
Scripps pier, La Jolla,
Calif.
E. Swift, 1965, South Atlantic, 730 miles east of
Rio de Janeiro
Culture Generation ~
..
. .
.
~ t a n a m g crop
¢ondttlonll
time
*, :[:
d~s§
4.5
mt-t
*, ¶
3.7
13,00011
*, ¶
4.3
50o011
450011
* Temperature 25 °C; Sylvania high yield cool white fluorescent lamps, 500 ft-c; LD 12:12 cycle;
no shaking.
:~Growth medium, Sweeney and Hastings (1957).
¶ Growth medium, Swift and Taylor (1967).
§ Under our conditions. In Jamaica, in nature, generation times of 2.5 days have been observed
II Approximately 21 days subsequent to inoculation at 500/ml.
used in the experiments. Fernbach flasks containing 1500 ml of media were inoculated
at 500/ml and grown into log phase without stirring. Concentrations were determined
by microscopic counting after gentle stirring which did not injure the organisms. 3
ml of the culture solutions were pipetted gently into 16 m m O.D. test tubes during
photophase and replaced in open wire racks in the growth chamber to insure recovery
from even this mild disturbance. Tubes were then removed from the racks without
jarring, for insertion into the bioluminescence assay geometries, at times depending
upon the properties being investigated.
B. Light Assay Techniques
All relative measurements of bioluminescence were made with E M I 9558 or E M I
6097 phototubes with cathodes at negative high voltage. Dynode resistor load currents, depending on phototube voltage, were of the order of 1 ma. Instantaneous anode
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currents never exceeded 0.01 m a and more usually were below 0.001 ma. Electrometer DC amplifiers (Seliger et al., 1962) were used throughout in conjunction with
Sanborn Model 320 recorders.
At this point we shall introduce a terminology to describe the various parameters
that were measured. All three species of dinoflagellates exhibit nocturnal stimulable
bioluminescence. The measurements of natural populations in Oyster Bay, J a m a i c a ,
W. I., were made with a photometer unit containing an impeller p u m p which stimulated the organisms to emit light and in addition maintained a constant flow of fresh
(a}
Chart Recorder
O. I/J.F
Anode of
Phototube
TSL
c
MS't
II
dt Channel I
"r. Channel 2
l"
iI
(b)
t0Lt09SZ
Anode of
Phototube /
Chart Recorder
SUSI
SUSI ~
Z Channel 1
I
Channel 2 ( X l O O g o i n )
FIGURe- 1. Schematic drawings of the feedback DC amplifiers used for measurement of
dinoflageUate bioluminescence. (a) Integrating circuit with RC differentiating network
at output of PC amplifier; (b) standard circuit for measuring light intensities.
organisms. Above some minimum pumping speed the observed mechanically stimulable
intensity, MSI, was direcdy proportional to the concentration of dinoflagellates being
pumped. In the laboratory measurements, a 3 ml volume of solution containing the
organisms was either stirred or bubbled until no further bioluminescence was emitted.
T h e total stimulable light, TSL, emitted by the sample was measured with the circuit
shown in Fig. 1 a. T h e mechanically stimulable intensity, MSI, as a function of time
was obtained with the differentiating circuit shown in Fig. 1 a. There is also a very
low-intensity bioluminescence observed in darkness under conditions of no external
stimulation. This spontaneous unstimulated intensity, SUSI, was measured with the circuit
shown in Fig. 1 b. T h e phototube gain and feedback resistors were set so that the
largest SUSI flashes could be recorded in recorder channel 1 without saturation,
while recorder channel 9, at up to 100 times the sensitivity of channel 1 (at a level
W. H. BIGGLEYET AL. Stimulableand SpontaneousBioluminescence
99
where the phototube dark current was just discernible) recorded small flashes not
detected in channel 1, as well as the continuous emission. In this way we could obtain
SUSI flashing rates and pulse height distributions simultaneously with the continuous
low-level SUSI. We also use L D 12" 12 to indicate a light-dark photoperiod of 12 hr
of light (photophase) followed by 12 hr of dark (scotophase). Times during these
phases will be referred to as D . or Ln. For example, De means 6 hr into the dark
period. The properties of bioluminescence that were examined as well as the time
periods involved are summarized in Table II.
TABLE
II
P A R A M E T E R S M E A S U R E D AND T I M E P E R I O D S OF M E A S U R E M E N T
Property of bioluminesccnccmeasured
Absolute photon emission per organism
Increase of M S I and TSL in dark
Constancy of M S I and TSL during dark
phase
Decrease of M S I and TSL in light
E n t r a i n e d r h y t h m of TSL
Entrained r h y t h m of S U S I
Diphasic character of TSL
Dark recovery of TSL
Time period of meaturcment
D4 to D8
L n to De
D1 to DI~
D6 to L6
D6 to D~o*
Do through D485
Placed in dark at L6; M S I and TSL assayed
subsequently
Stimulated at D6; M S I and TSL assayed subsequently at 5 rain intervals
* D20 means t h a t the lights were not turned on at DI~ but that the organisms were continued
in darkness for 8 more hr.
D48 means continued darkness for 36 hr past DIs.
C. Mechanical Stimulation of Organisms
T h e dinoflagellates were mechanically stimulated by both bubbling and stirring. T h e
sample test tube containing 3 ml of dinoflagellates in culture medium was fixed
vertically and reproducibly alongside the end-on phototube face. Stirring was
accomplished with the " U " portion of a straightened Giant Gem paper clip, fitted into
the rotor of an 1800 rpm motor. T h e U portion of the clip extended approximately
two-thirds of the way into the sample and rotation produced an extremely turbulent
solution. Bubbling was produced by means of a small air pump adjusted to a flow of
1.5 liters per min through a No. 15 cannula (0.137 mm I.D.), with the cannula tip
extending halfway down into the sample.
D. Spontaneous Unstimulated Intensity
Samples of SUSI were measured as a thin layer (6 mm) of solution in a flat-bottomed
quartz cylindrical cell (2.5 cm I.D. (inside diameter)) sitting directly on the flat face
(5 cm D) of a vertically mounted phototube. The cell was covered by a hemispherical
mirror to increase the efficiency of the light collection and also to retard evaporation
during the measuring period (see Table II). In this way the geometry was reasonably
constant for all organisms emitting within the volume.
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RESULTS
All data are reported in absolute units of photons per second per organism
for intensities (MSI, SUSI) or photons per organism for total light (TSL),
based on an independent absolute calibration reported separately?
A. Experimental Precision
There are several sources of error in the determination of TSL per organism.
There are (a) possible nonuniformities in organism concentrations in the
culture flasks; (b) real differences in TSL per organism in any particular
species, depending on the age and density of the culture, the previous lightdark history of the culture, the natural variability of the organisms themselves, and differences among different cultures of the same species; (c) changes
over long periods of time, in either spectral sensitivity or amplification factors
of the phototubes used for light measurements; (d) variations in the degree of
turbulence produced by the mechanical stimulation method.
For (a) we have been able to demonstrate that with gentle stirring in the 1
liter culture flasks, successive withdrawals of 1 ml samples for nficroscopic
counting could be counted with a coefficient of variation s of 6 %.
For (b) we have shown that for all three species T S L is constant from approximately D1 through Dn within the precision of a set of measurements
made over a short time interval. The coefficient of variation of TSL for a
series of 20 consecutive samples of G. polyedra was 11%.
We have followed individual cultures from the time of inoculation through
log phase of growth into stationary phase, in some cases for as long as 27 days.
Typical data for T S L per organism determined at D4 for all three species are
shown in Table III. Since we have not yet studied the effects of intensity and
the spectral quality of ambient light on the growth and bioluminescence of
these organisms, we maintained our culture conditions constant so that at
least the species comparisons could be made. We did verify, however, that a
factor of two increase in ambient light intensity had no effect on the observed
bioluminescence.
For (c) we used a hermetically sealed, radioactive light source, consisting of
~4C homogeneously incorporated into a luminescent epoxy resin. With this
constant intensity light source we have been able to maintain a continuing
check on our phototube sensitivities.
a Seliger, H. H . , W . H. Biggley, a n d E. Swift. 1969. T o t a l stimulable light emission in t h e m a r i n e
dinoflagellates, Pyrodinium bahamense, Gonyaulax polyedra and Pyrocystis lunula. Photochem. Photobiol. I n
pre~.
2 Defined as t h e s t a n d a r d deviation divided by t h e m e a n in per cent.
W. H. BIGGLEY ET AL.
Stimulable and Spontaneous Bioluminescence
ioi
For (d) we verified that changes of 4-50 % in the bubbling rate or the
stirring rate did not affect the T S L readings during scotophase.
B. Photoperiod-EntrainedRhythms of Nocturnal TSL per Organism
1. LABORATORYCULTURES
None of the three species exhibits exactly the same kinetics of MSI. For
example the MSI of P. bahamense during photophase and subsequent to the
TABLE
III
E X A M P L E S OF E X P E R I M E N T A L P R E C I S I O N F O R L A B O R A T O R Y C U L T U R E S
T H R O U G H O U T L O G PHASE G R O W T H
P. bahamense
T i m e after
inoculaCells in 3 ml
TSL/cell
tion
days
4
5
6
7
8
11
12
13
14
15
18
20
22
25
27
G. polyedra
Cells in 3 ml
860
3.30
2,810
1,090
1,350
1,000
2,440
2,840
3,390
2.72
3.19
3.63
3.92
3.00
3.55
3,480
6,570
6,830
13,440
12,220
12,700
4.78
3.55
3.92
2.29
3.00
2.73
4 020
6000
9000
10 350
16 620
17 840
22 190
23. 140
32 150
38 410
38,280
40,020
50,810
Coefficient of variation :
17°/o
Ceils in 3 ml
1.26
1.24
1.21
1.05
1.20
1.01
1.20
1.10
1.15
1.14
1.03
1.27
1.34
1.19
TSL
Average cell : 1.17 X 108
Coefficient of variation :
8.2°/o
TSL/cell
photons X 10-8
photons X 10 -8
photons X 10 4
TSL
Average cell : 3.35 X l0 s
TSL/cell
P. lunula
1,520
2,040
2,040
2 520
2 910
5 570
4 910
5 520
6 920
74OO
9 270
9 220
9 960
12 010
20.2
45.6
51.0
39.2
46.5
34.9
47.7
40.4
36.5
35.6
35.6
36.5
37.1
38.1
TSL
Average cell :38.9 X l0 s
Coefficient of variation :
19%
onset of scotophase is shown in Fig. 2. Mechanical stimulation of a population
of P. bahamense results in a cumulative flash which has the same relative shape in
scotophase as in photophase.
T h e M S I flashes of P. lunula are essentially the same as those of P. bahamense.
However, as the result of mechanical stimulation, P. lunula emits a low-level
continuous glow, presumably due to cell damage, which persists after mechanical stimulation has stopped. A glow is also observed if a droplet of culture
medium containing the organisms adheres to the side of the test tube and the
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solution begins to e v a p o r a t e . F o r this reason, o n l y the p a p e r clip stirring
t e c h n i q u e , w h i c h e v o k e d the m a x i m u m initial M S I a n d resulted in the least
splashing of solution to the sides o f the test tubes, was used for T S L m e a s u r e -
MSI P.
bahomense
"~.~-20-68
STIMULATION BY STIRRING
o"
D+3#
o3
F]om~a~ 2. Shapes of MSI
flashes of P. bahamense populations during the transition from
photophase to seotophase. The
ordinates are in relative intensity units for each curve. The
curves are displaced vertically
from one another by distances
corresponding to the time
during scotophase at which
MSI was measured. The three
vertical sections were measured
at relative electronic gains of 1,
1/10, and 1/100.
5:
~>
n~
1:)+5'
D+O'-
D-18'~l = i J
0125456
Seconds
Seconds
Seconds
m e n t s o f P. lunula. D u r i n g p h o t o p h a s e the c o n t i n u o u s g l o w e m i t t e d as the
result o f m e c h a n i c a l s t i m u l a t i o n p r o d u c e d a m u c h l a r g e r c o n t r i b u t i o n to
T S L t h a n the M S I flash. W e a s s u m e d t h a t this was d u e to i n j u r y to t h e or-
W. H. BIGGLEYE,T AL. Stimglabl¢and Spontaneous Bioluminescence
zo3
ganisms. Therefore for all TSL measurements of P. lunula we arbitrarily integrated the light emission over the time period during which the flash decays
to 10 % of its peak value. During scotophase, the glow induced by mechanical
MSI G.
polyedro
"v'- 20 - 6 0
STIMULATION BY STIRRING
O
D+7~
"-.-
w>=
D+3G
D+ 1 9 ' / ~
D+I2'
D+ 7'.
D÷ O'
D_3',F
o
~
~
,b ,~ ~'o 2'5 ~'o ~5 ~o ~
Seconds
345
~o 61~"'
Seconds
~
o l23k
Sec~ds
FIOURE 3. Shapes of MSI flashes of G. polyedra populations during the transition from
photophase to scotophase. The ordinates are in relative intensity units for each curve.
The curves are displaced vertically from one another by distances corresponding to the
time during scotophase at which MSI was measured. The three vertical sections were
measured at relative electronic gains of 1, 1/10, and 1/100.
stimulation was negligible compared with the M S I flash and therefore the
time period over which T S L was measured was not as critical.
G. polyedra does not exhibit this stimulation-induced glow. However, during
photophase there are two different types of mechanically stimulable bio-
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luminescence (Fig. 3). There is a low-level continuous MSI component which
can persist for several minutes. This component is extremely sensitive to the
degree of turbulence produced by the stimulation technique and is much less
intense for bubbling (even under our optimum conditions) than for stirring.
Around Do + I0 min MSI flashes become apparent and the low-level continuous MSI decreases. Subsequent to Do + 30 min there is no observable
low-level continuous MSI, and the MSI flashes of G. polyedra have the same
relative shapes as those for P. bahamense and P. lunula. However, even in
P.bohomense
TSL
IO
X-10-67
._,l
O-STIMULATION
BY BUBBLING
• -STIMULATION
BY STIRRING ~ - - Q = r ~ , - . - ~ c J
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FIOUm~ 4. Photoperiod-entrained T S L per organism for P. bahamense in absolute units
of photons over a complete photoperiod.
scotophase, T S L of G. polyedra was more sensitive than that of the other two
species to degree of turbulence, and T S L values obtained with paper clip
stirring were consistently higher than those obtained with bubbling.
Single periods of the photoperiod-entrained rhythms of nocturnal T S L per
organism in absolute units of quanta for P. bahamense, G. polyedra, and P.
lunula are shown in Figs. 4-6, respectively. It should be emphasized that the
data of Figs. 4-6 are repeatable during every photoperiod over the entire
culture life (inoculation through stationary phase) as implied by Table III.
Because of the large scotophase to photophase variations, the T S L data are
plotted on a logarithmic scale. The abscissae are in hours relative to the beginning of scotophase or photophase, which in laboratory cultures are step
functions. The scotophase is shaded. In Fig. 4, the results of bubbling and
!
L6
W. H. BIOOLEYET AL. Stimulableand SpontaneousBioluminescence
xo5
paper clip stirring for P. bahamense are plotted together since T S L was equally
sensitive to either method of stimulation.
T h e paper clip stirring data for G. polyedra are plotted in Fig. 5 a separately
from the bubbling data which are shown in Fig. 5 b. As we have described
above, T S L during scotophase (D, to Dn) is the result of a large M S I flash
which decreases to zero intensity within approximately 5 see (Fig. 3). Thus
T S L is independent of the integration time. However, during photophase, the
integration time determines the total light measured. In Fig. 5 a values of
T S L per organism during photophase are shown for integration times of 5, 30,
and 90 see. T h e large differences between bubbling and stirring T S L during
photophase for G. polyedra are evident from Fig. 5 b in which for the same culture, T S L per organism is plotted for bubbling periods of 120 see. Even during
scotophase, T S L per organism obtained by bubbling is somewhat lower (15 %)
than that obtained by stirring.
T h e corresponding data for stirring T S L per organism for P. lunula, shown
in Fig. 6, agreed with checking experiments in which bubbling was used for
stimulation, although as explained above, the bubbling data were more
erratic because of splashing.
For ease of discussion, we shall list the following points: (a) T h e increases
in T S L per organism subsequent to D 0 are extremely rapid and exponential,
with doubling times of 5-8 rain for all three species. (b) M a x i m u m values of
T S L per organism are attained within 60-90 min of initiation of scotophase.
(c) From D, through Dn, T S L per organism is constant within the precision
of the measurements. (d) There is a small decrease in T S L per organism between D n and D,2, just before the initiation of photophase. T h e effect is
largest for G. polyedra. (e) Subsequent to photophase, there are rapid exponential decreases in T S L per organism. However, under our laboratory growth
conditions, the decrease for P. bahamense (Fig. 4) is not nearly as rapid as for
G. polyedra or P. lunula. T h e half-times for decrease of T S L per organism are
50 min for P. bahamense, 9 min for G. polyedra, and 4-5 min for P. lunula. (f) The
m a x i m u m scotophase-to-photophase ratios of T S L per organism are approximately 200:1 for P. bahamense and G. polyedra. For P. lunula the ratio is m u c h
larger and depends on the integration time during photophase. If we arbitrarily integrate over the time during which the photophase flash decays to
10 % of its peak value, the ratio is 950: 1.
2. NATURAL POPULATIONS
We performed similar measurements of T S L per organism with natural populations of P. bahamense, freshly removed from Oyster Bay, Jamaica, W. I.
Samples taken during natural photophase were separated into two sets of test
tubes. Set A was placed in total darkness one-half hour before sunset. Set B
was allowed to remain in natural photoperiod. T S L per organism was meas-
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T H E
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P H Y S I O L O G Y
•
V O L U M E
54
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I969
u r e d for b o t h sets at intervals t h r o u g h o u t the next 19 hr. W e o b t a i n e d the following results: (a) T h e i m m e d i a t e rate of increase in T S L per organism for
set A (step function scotophase) was greater t h a n for set B (natural scoto1 0 9 --_
G. po lyedra
~-20-67
STIMULATION BY STIRRING
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FmURE 5 a
FIGURE5. Photoperiod-entrained TSL per organism for G. poly¢dra in absolute units of
photons over a complete photoperiod. (a), TSL per organism as a result of paper clip
stirring. During photophase and the transition time Do to D1 light emission was integrated
for 5, 30, and 90 sec as shown by the encircled numbers. The data during scotophasc
represented bythe solid circles are independent of time of integration. (b), TSL per organism as a result of bubbling. During photophase and the transition time Do to DI light
emission was integrated for 120 sec. During scotophase the data are independent of time
of integration.
phase). (b) F r o m 1 hr past sunset t h r o u g h o u t the scotophase, T S L per organism was identical for both sets. (c) Subsequent to dawn, set B (natural photoperiod) exhibited the same rapid decreases in T S L per organism as the M S I
n a t u r a l photoperiod d a t a of Seliger a n d M c E l r o y (1968). (d) Set A, kept in
darkness t h r o u g h D17, exhibited a very g r a d u a l decrease in T S L per organism
beginning a r o u n d Du. (e) At D17, corresponding to L5 of the n a t u r a l photo-
Stimulableand SpontaneousBioluminescence
W . H . BXGGLEY ET AL.
xo7
period, set A was removed from the dark and placed in ambient sunlight. The
decrease in TSL per organism was precipitous, even more rapid than observed
for set B under natural photoperiod.
[0
9
G.polyedro
--
X-10-67
STIMULATION
n~
BY BUBBLING
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,
l
DI 2
L2
L4
PHOTOPHASE
FIGURE 5 b
3.
COMPARISONS OF TSL P E R ORGANISM
Circadian rhythms of TSL have been reported for G. polyedra (Hastings and
Sweeney, 1957), for P. lunula (Swift, 1967), and for a heterogeneous natural
population (Kelly and Katona, 1966). These rhythms were observed under
constant external conditions of illumination, either continuous dim light or
continued darkness, as differentiated from the photoperiod-entrained rhythms
of TSL. In captured natural samples of P. bahamense kept in continued darkness we have observed higher values of TSL at times corresponding to "night"
and lower values of TSL at times corresponding to "day." In continued
darkness the magnitudes of the TSL values were damped completely after 72
hr (see also Fig. 1 of Kelly and Katona, 1966). Microscopic observation
lO8
THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 54 "
x969
showed that the damping of the T S L values was due to death of the organisms
in continued darkness. Laboratory cultures of all three species grown into
log phase under LD 12:12 photoperiod will be essentially killed after 72 hr of
extended darkness. Laboratory cultures of P. bahamense are the most sensitive
to extended darkness; they are killed within 48 hr. With this limitation placed
on the observations we have verified that for laboratory cultures of P. bahai0
I°
_
STIMULATION
BY
STIRRING
m
•
@
.-1
/
LI.I
0
io
(E
L[.I
0..
9
_
TSL
P. lunula
v -?_1-67
O3
Z
0
I-0
-tO_
I0
8
E
:E
O3
Z
(.9
nO
a:
ILl
EL
I0
7
.-I
O3
I--
@
I0
i
L4
[
i
L6
i
f
Le
PHOTOPHASE
i
!
LIO
i
i
DO
i
i
D2
i
i
D4
i
i
D6
i
i
Ds
SCOTOPHASE
i
Dio
i
D=2
i
Lz
i
L4
!
Ls
PHOTOPHASE
FiGtra~, 6. Photoperiod-entrained TSL per organism for P. lunula in absolute units of
photons over a complete photoperiod.
mense in continued darkness there are: an increase in T S L during the interval
D 0 to Du, a decrease in T S L during Du to D24, an increase in T S L during D24
to D86, and a decrease in T S L during D36 to D~8, corresponding to a highly
damped oscillation. For G. polyedra and P. lunula the damping is less extreme
and permits the observations of T S L periodicity for an additional 24 hr. In
all cases the damping is due to death of the organism. Although, as one of the
reviewers has pointed out, these observations of periodicity do not extend
over an appreciable length of time, we feel that this suggests the presence of
an endogenous circadian r h y t h m of T S L in P. baharnense.
W. I-'I BTGGLEYET AL. Stimulableand SpontaneousBioluminesceme
xo9
We further observed for all three species that during the time interval D12
to D1 s the dinoflagellates did not die and the rates of decrease of T S L per
organism for cultures in extended darkness were markedly different than the
rates of decrease of T S L per organism for cultures in normal LD 12: 12 photoperiod (for these latter organisms the comparable time interval was L0 to Le).
The comparisons of these two rates for all three species are shown in Fig. 7.
-_
P.lunula
JO I0 ---
._J
._]
LU
0
~- 109
03
z
0
I0
r-la_
~
--=--'=~i'~.A
I
I G.po/.vedra
(a)
-
P.
bahamense
(b)
I0 e
\
7
0 I0 7
¢'r
Ixl
A
/,
._J
t
I--- i0 6
I0 ~
+
--
I
,I
I
I
qo
qz
LZ
L4
Scotophase
Photophase
I
L6
~
+
t
Dlo
Dtz
Lz
L4
Scotophase Photophase
D~o
I
Diz
Scotophose
I
Lz
I
L4
I
Ls
Photophase
FiougE 7. a, decrease in TSL per organism for laboratory cultures of P. lunula subsequent to initiation of photophase, compared with TSL per organism in continued darkness. Open triangles, photophase; solid triangles, continued darkness. 6, decrease in TSL
per organism for laboratory cultures of G. polydra subsequent to initiation of photophase,
compared with TSL per organism in continued darkness. Open triangles, photophase,
30 see paper clip stirring; open circles, photophase, 120 sec bubbling; solid triangles,
paper clip stirring in continued darkness; solid circles, bubbling in continued darkness.
c, decrease in TSL per organism for laboratory cultures of P. bahamensesubsequent to
initiation of photophase, compared with TSL per organism in continued darkness. + ,
Normal photophase, 500 ft-; open triangles, photophase, 2000 ft-; solid triangles, continued darkness.
I
L8
fig
THE JOURNAL
OF GENERAL
PHYSIOLOGY
• VOLUME
54
"
I969
Due to the fact that the organisms remain viable at least through Dis, the
data of Fig. 7 present a valid comparison of the effect of light on the reduction
of TSL. The half-times for both scotophase- and photophase-induced changes
in TSL per organism for all three species under the different experimental
conditions are summarized in Table IV.
We set up a separate culture of P. bahamense in natural Baltimore photoperiod (2500 ft-c at noon) in an effort to approach the spectral quality and
intensity of ambient sunlight in Jamaica. As shown in Fig. 8, even under these
conditions natural photophase TSL for P. bahamense remained essentially unchanged from that measured for fluorescent light photophase. We have, howTABLE
HALF-TIMES
FOR
PHOTOPHASE-INDUCED
IV
SCOTOPHASEAND
CHANGES
IN TSL
Laboratory photoperiod
Species
Increase in
Decrease in
TSL subsequent TSL subsequent
to step-function to step-function
laboratory
laboratory
scotophase
photophase
Extended
darkness
Natural photoperiod
Increase in
Decrease in
Decrease in
Decrease in
TSL during TSL subsequent TSL subsequent TSL subsequent
natural
to natural
to natural
to Dlz in
Jamaica
Jamaica
Baltimore
continued
scotophase
photophase
photophase
darkneB
min
min
min
min
min
min
P. bahamense
5-8
40-50
6-10
10-20
54*
G. polyedra
P. lunula
7
5
6-9
4-5
---
---
---
160i~
330*
70~
180i~
* Laboratory
Laboratory
c u l t u r e g r o w n i n n a t u r a l p h o t o p e r i o d ( B a l t i m o r e s u n l i g h t , 2500 ft-c, n o o n ) .
c u l t u r e g r o w n i n f l u o r e s c e n t l i g h t L D 12:12 c y c l e (500 f t - c ) .
ever, been able to produce an extremely rapid decrease in TSL per organism
for a laboratory culture of P. bahamense if at L 0 the culture is placed for only
10-15 min at 17 cm from a 1000 w high pressure mercury arc (5000 ft-c).
C. Kinetics of M S I in Photophase and Scotophase
The shapes of MSI during the transition from photophase to scotophase have
already been described. The question now arises as to what happens to MSI
if the lights are not turned on subsequent to Du? Does the organism exhibit a
gradually reduced "scotophase" MSI or are the kinetics of MSI those of
"photophase"? Figs. 9 and 10 are direct tracings of curves from the chart of
the two-channel recorder of Fig. 1 a, for P. bahamense and G. polyedra, respectively, for representative times during scotophase, photophase, and for the organisms continued in the dark past Dt~. The electronic gains for measurements during these different time periods were quite different from one
another and the curves represent only the shapes of the MSI flashes and the
W. H. BIOGLEYEr M.. Stimulableand SpontaneousBioluminescence
III
integrated light intensities. Both P. bahamense and P. lunula exhibit the same
kinetics of stimulable bioluminescence, independent of phase of photoperiod.
In the case of G. polyedra, we have presented side-by-side comparisons of
stirring and bubbling data, the latter taken at the same electronic gain with a
fresh sample within 2 rain of the stirring measurement. During scotophase
(Fig. 10 a) both methods of stimulation give essentially the same M S I flash
10 9
g
107 --
n~
0
i0 ~
•" -
,.
-"--
~a
~:
m
z
_j
_
~
CO
~
i,i
O..
P, bohomense
o
LABORATORY
BALTIMORE
LABORATORY
CONTINUED
I
0500
1
3ZI.-4-6B
•
I-",I tv3 5
04.00
CULTURE
PHOTOPHASE
I
0600
I
0700
U
0800
TIME
CULTURE1
DARK
I
I
0900
I000
I
II00
I ,
1200
,I
1300
OF DAY
FxougE 8. TSL per organism for laboratory cultures of P. bahamenseduring natural
photophase compared with organisms remaining in constant darkness. Open circles, organisms in natural photophase in Baltimore (sunny day); solid circles, organisms kept
in continuing darkness.
kinetics, although the bubbling results are always slightly lower than those
for stirring. T h e double peak in the M S I flash is a stirring artifact. It is during
photophase (Fig. 10 b) that the marked difference in stimulability arises,
giving rise to the concept of two different types of mechanically stimulable
bioluminescence.
In all three cases, during extended scotophase or continued darkness, the
kinetics of stimulable bioluminescence are essentially those of the normal
scotophase.
II2
THE
JOURNAL OF GENERAL PHYSIOLOGY • VOLUME
54 • t969
D. Spontaneous Unstimulated Intensity (SUS1)
Hastings and Sweeney (1959) reported an endogenous circadian r h y t h m of
SUSI for G. polyedra in continued darkness and in constant dim light. With
our present equipment, we have been able to look at some of the details of
___•5F.,•.•
~. bohomense TSL
D +10::30
P.bohamense TSL
, 5 sec i
b
~r
L + :3
RbahomenseTSL
:~ec'~
D--~I5 ; 38"
stir
SCOTOPHASE
stir
PHOTOPHASE
stir
DARK
FxotmE 9. Direct tracings of
chart records of TSL and MSI
for P. bahamensefor representative times during a), scotophase,
b), photophase, and c, continuing darkness past DI~. The
electronic gains for a, b, and c
were different and the curves
show only relative shapes.
this SUSI. For P. bahamense, G. polyedra, and P. lunula, SUSI consists, to different degrees, of a low-level continuous emission which is proportional to cell
concentration, individual small flashes from single cells, and occasional
transient glows lasting for several seconds.
In Section B 3 we have referred to the rapid death of the dinoflagellates
under conditions of extended darkness. This applies as well to measurements
of SUSI in extended darkness.
G. polyedra, as expected, exhibits an endogenous r h y t h m of SUSI in com-
W. H. BIc.oLEY ~.T ~ .
Stimulable and Spontaneous Bioluminescenee
i[3
plete darkness. Any periodicity in S U S I for P. bahamense or P. lunula is apparently so highly d a m p e d that it is not observable except for the initial scotophase time interval. Comparable data for S U S ! for G. polyedra, P. bahamense,
and P. lunula are shown in Fig. 11 a, b, and c. S U S I is plotted as the n u m b e r
~_~
5sec
',:
G.polyedroTSL
stir
-~,
SCOTOPHASE
O---+'~: 4-7
~55
1~
G.polyedroTSL
sec
=I
Cl
G.polyedroMSI
D+10:48
G.polyedroMS 1
stir
bubble
SCOTOPHASE
bubble
L
t / '~/Reset
]
Capacitor
.
G.polyedro TSL bubble
1-
f
C
:Ssec
"[
L +4:05
G . p o ~ e ~ o TSL
D+16:02
G..po~edroMSZ
PHOTOPHASE
stir
DARK
stir
"~,5
,-
sec ,
-,
L+4:07
G.polyedroTSL
G.polyedroMs'r
PHOTOPHASE
bubble
"=bsec ! D + 1 6 : 0 4
DARK
bubble
FIotmJz 10. Direct tracings of chart records of TSL and MSI for G. polyedra for representative times during a, scotophase, b, photophase, and c, continuing darkness past Dx~.
The side-by-side stirring and bubbling curves can be compared directly. The electronic
gains for a, b, and c were different and the curves show only relative shapes. In b, MSI
was so small that it is not shown.
of photons per organism per second, averaged over a 5 m i n period, measured
at 15 m i n intervals from D 0 through D48 (36 hr of darkness past DI~.).
S U S I for G. polyedra (Fig. I I b) is predominantly a glow a n d can be divided
into three parts: (a) A low-level constant portion from Do to Dx0 where the
average rate of light emission per organism is 280 photons per second; (b) a
narrow peak at D n with a ha[f-width of 100 m i n and a m a x i m u m intensity
per organism of 1500 photons per second; (c) a subsequent decrease to intensities lower than (a) from Dx8 through D~8 at which time (a) and (b) are
repeated.
II 4
THE
JOURNAL
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PHYSIOLOGY
•
54
VOLUME
" I969
E. Dark Recovery of Stimulable Bioluminescence
During scotophase all three species, after having been stimulated to exhaustion, can recover part of their initial bioluminescent capacity within a short
3000
2500
P, bahamense
200C
150C
A
m ]ooc
L0
50C
CD
O~
o
o
2ooo~
I~
tsoo/
G. polyedra
I I
f,,;IT
I
Z
O
-r
o-5000
_ 4500
O9
4000
O3
P. l u n u l o
(c)
IO0C
Vo
6
12
18
24
50
56
42
48
HOURS IN CONTINUED DARK
F I G U ~ 11.
SUSI in photons per second per organism beginning at scotophase, Do, and
continued in darkness through D~. a, P. bahamense. O p e n circles, total SUSI, flashes
plus glow; heavy line, glow only. b, G. polyedra. O p e n triangles, flashes only; heavy line,
glow only. c, P. lunula. O p e n circles, total SUSI, flashes plus glow; heavy line, glow only.
time. This process can be repeated several times, although each recovery
results in progressively smaller values of TSL; this is especially true for P.
lunula, presumably due to cell damage during stimulation. Recovery data are
plotted for all three species in Fig. 12, as per cent of initial TSL as a function
of time subsequent to stimulation. The recovery is exponential with half-
W.
H.
Slimgtobl8 aM
B I G O L E Y E T AL.
SpontaneousBioluminescenee
II 5
times of 18, 20, and 30 min for P. bahamense, G. polyedra, and P. lunula, respectively.
F. Spectral Intensity Effects and Phase Setting
A natural population was removed from Oyster Bay in J a m a i c a during natural
photophase and placed in complete and continuous darkness. For the data of
I00
x-x-~
80
60
O n
x/x
: y
40
"%xSe*
G. polyedra
• stimulation by stirring
x stimulation by bubbling
?t
20
t
0
0
co
I
I
I
I
20
40
60
80
~ IO0
o
x
X~X
~o. 8o
I
I
I00
120
MINUTES
I
I
I
140
160
180
~
X
200
x
"*" X
X
x xxA/
X
.__._L_;
•
~
4O
P. bohomonse
= stimulation by stirring
x stimulation by bubbling
20
z
w
0
f
f
I
f
20
40
60
80
I
I
I00
120
MINUTES
I
I
I
140
160
180
200
a~
4O
P. /unu/o
30
stimulation by stirring
~°I
2O
0
•
20
40
o _ 1 . _ _ ~ ~
-----e
I
I
I
I
I
I
I
60
80
I00
120
140
160
180
200
MINUTES
FlouU.~ 12. Recovery of stimulable biohnninescencefor G. polyedra, P. bahamense,and P.
lunula. Time is measured subsequent to complete stimulation by the technique used to
measure recovery.
Fig. 13 (open circles), this was done at 13:12, approximately the middle of
natural photophase, although the time is not critical. T h e r e was an immediate
rapid increase in T S L reaching a value greater by a factor of approximately
40 within an hour, at which time a plateau was reached. T h e plateau was
maintained until approximately the time of day at which the in situ P. bahamense began their nocturnal increase in T S L per organism. At this time the
~6
THE
JOURNAL
OF
OEN~I~AL
I~HYSIOLOOY
• VOLUM~
54
"
"N
O
"
~ V
,~
O
O
0
O
0
0
O.~
~
I.-
0
v
0
~ ,~
V- .o
"~'
0
0
0
..
f,,.
/,0
I
0
0
OJ
I
v
n"
SS
0
0
0
Ij]11t
%
I
I
1111111
"o
INSINVg~O
I
|
IIIl111
I
I
%
~13cI
ISIN
~0
Illllll
%
7S1
I
I
I1|1111
o
3AIIV73~I
~
~-
o
~
~
~969
W. H. BIC,GI~P.v ~.z AL. Stimulableand SpontaneousBioluminescence
II7
population confined in the dark exhibited a second rapid and exponential
increase in T S L per organism, as though scotophase had just begun. For
comparison the relative M S I data for the in situ bay population, obtained
over the same time interval with the underwater photometers described by
IO 9 - -
P bohctmense
,_1
--I
I.LI
=, g . . . -
(,0
Z
o I0 e
n0.
(J)
z
(p
Dz I 0 7 - 0
a.
_J
or)
o o
o
I0
I
I
L4
I
Y
Le
f°l
~
L8
PH OTO PHASE
°
Lm
d
I
DO
I
Dz
I
I
D4
SCOTOPHASE
Fxoul~ 14. TSL per organism for laboratory cultures of P. bahamense as a function of
photoperiod time. The cultures were removed from the fluorescentlight photophase and
placed immediately in continued darkness. For comparison, we have also plotted the
normal photoperiod-entrained rise in TSL per organism. Solid circles, cultures removed
from photophase at Le; solid triangles, cultures removed from photophase at Lg; open
circles, control removed from photophase at LI~.
Seliger and McElroy (1968), are shown as the solid triangles. T h e nocturnal
increase in T S L per organism for the sample confined in the dark remains in
phase with the natural photoperiod and thus is independent of the time during
photophase at which the dinoflagellates are artifically placed in the dark.
T h e initial dark-induced rise is immediate. T h e duration of the initial
plateau is dependent upon the time during natural photoperiod when the
organisms are placed in the dark. In nature, the two effects cannot be separated.
T h e same experiments repeated with laboratory cultures of P. bahamense,
placed in darkness at L6 and at L9 (Fig. 14), showed a gradual rise to a maxim u m value of T S L per organism at D1.
II8
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
54
"
1969
DISCUSSION
Under carefully controlled conditions of organism growth and with precisely
defined stimulation and bioluminescence assay techniques, it has been possible to describe and compare several characteristics of the bioluminescence of
three species of dinoflagellates. These characteristics such as the changes with
time of the MSI flash, the T S L per organism, and SUSI are indicative of the
physiological state of the organisms and should provide a sensitive assay
technique for observing the effects of physical and chemical perturbants on
the organisms.
Although in nature it has been our experience that dinoflagellates are
seldom maximally stimulated mechanically to emit all of their potential
luminescence, we have been able to demonstrate that they can be maximally
stimulated without preventing further growth of the culture. This potential
for stimulable luminescence, although different for different species, is a constant throughout essentially the entire scotophase (D1 through Dn), in agreement with the data of Seliger and McElroy (1968) for natural populations of
P. bahamense. The data presented are not intended primarily to demonstrate
the existence of photoperiod-entrained and endogenous rhythms of bioluminescence; for the most part these have already been established by many
previous workers. Rather we have examined the detailed shapes of the bioluminescent emissions from three species over their photoperiods as well as in
extended darkness. For the photoperiod-entrained rhythm there are extremely rapid increases and decreases in T S L per organism (Table IV), so
that for a complete photoperiod the stimulable bioluminescence has essentially a square-wave shape (Figs. 4-6). These shapes are completely different
from the sine wave shapes reported by previous workers. In extended darkness, however, the endogenous rhythm of T S L does exhibit a sine wave
character as described by Hastings and Sweeney (1957), Kelly and Katona
(1966), and Swift (1967). However, since extended darkness for even 19-24
hr results in permanent injury to the dinoflagellates, we have limited our
comparisons to T S L to the first 6 hr (Du through D~8) of extended darkness.
From the data of Fig. 7 and the last column of Table IV, it may be seen that
the rates of decrease of endogenous T S L are different for the three species,
over and above the fact that these rates are each significantly lower than their
respective photoinhibited rates of decrease during photophase. For G. polyedra
and P. lunula our laboratory photophase intensities are sufficient to achieve a
m a x i m u m photoinhibition, as evidenced by the rate of decrease of TSL. For
the tropical P. bahamense a much higher light intensity is required. There are
two aspects of the photoinhibition during photophase that must be examined :
(a) the rate of decrease of T S L should have a spectral intensity dependence; (b)
MSI (scotophase)
from the data of Fig. 13 and the fact that, for P. bahamense, the
MSI (photophase)
W. H. BIGOLEY ET AL. Stimulable and Spontaneous Bioluminescence
II9
ratios in nature, in J a m a i c a sunlight, are higher than those observed for our
Scotophase TSL
laboratory cultures indicates that the Photophase TSL ratios as well should exhibit
a spectral intensity dependence.
We have been able to achieve a rate of decrease in T S L per P. bahamense as
fast as that for P. lunula by placing the P. bahamense culture close to a high
intensity 1000 w mercury arc for as little as 10 min at L0. However, we have
also observed that transitory morphological changes in pigment distributions
occur when dinoflagellates are subjected to light intensities higher than those
occurring during their normal photophase. For this reason all our measurements with the exception of the H g arc illumination have been performed on
cultures inoculated and grown up into log phase under the same spectral
intensity and photoperiod as were used in the experiments.
Since of the three species, P. bahamense is the only representative of shallow
tropical waters, it is not unreasonable to believe that the light intensities of our
laboratory environmental chambers and even Baltimore photophase are not
sufficient to effect the rapid T S L decrease observed in J a m a i c a sunlight. In
laboratory culture, photophase is 500 ft-c. In Jamaica, sunlight illumination
is as high as 8800 ft-c.
Nothing is known at present of the mechanism by which mechanical stimulation triggers light emission.
As in the case of the marine bacteria, there does not appear to be any
selective advantage to the bioluminescence of dinoflagellates. In nature nonluminous species are often found together with luminous species (Nordli,
1957).
T h e significant differences in stimulability during photophase between P.
bahamense and G. polyedra, which are responsible for the bubbling vs. stirring
differences shown in Fig. 5, are demonstrated in Figs. 2 and 3 as well as in
Figs. 9 b and 10 b. There are also differences in degree of recovery of potential
for stimulable bioluminescence among all three species (Fig. 12).
All three species show an increase in SUSI subsequent to initiation of scotophase. P. bahamense and P. lunula show large fluctuations in SUSI over D 0 to
D u (mainly due to erratic flashing) and then decrease to low levels. O f the
three species, only G. polyedra exhibits well-defined peaks in both SUSI "glow"
and cell division (Sweeney and Hastings, 1958) as functions of photoperiod.
T h e former occurs at D n and has a half-width of only 100 rain, while the
latter occurs at L1 with an even smaller half-width of 70 min. 3 For all three
species the PC glow, represented by the heavy lines, is m u c h less erratic than
the flashes. For G. polyedra as contrasted with P. bahamense and P. lunula the
contribution of flashes to SUSI was so small that in Fig. 11 b it is plotted
separately. T h e second peak in SUSI glow around Dae is the expression of the
circadian rhythm.
3 Unpublished data.
12o
THE JOURNAL
OF G E N E R A L
PHYSIOLOGY
• VOLUME
54
" 1969
T h e distinction between glow or DC level and a large n u m b e r of tiny
flashes depends upon the signal-to-noise ratio of the phototube detector and
the frequency response of the associated electronics. In our case the phototube
dark noise was equivalent to an average dinoflagellate emission of 1.3 X l0 s
photons per sec. T h e input R C time constant of the DC amplifier was 0.01
sec. We arbitrarily set our "recognition" of flashes to those which, during any
0.01 sec time interval, emit a n u m b e r of photons equal to the average n u m b e r
of "noise" photons; i.e., bioluminescent flashes emitting 1300 photons within
0.01 sec. Since the mean lifetime of the decay of dinoflagellate flashes is 0.05
sec, this corresponds to flashes containing > 6500 photons. Thus under ideal
conditions SUSI flashes emitting approximately 10-5 of the scotophase MSI
per organism for P. bahamense and G. polyedra and 10-6 of the scotophase M S I
per organism for P. lunula could be detected.
We have not yet examined the effects of light intensity, temperature,
nutrient levels, etc. on SUSI for these species and so we cannot say with certainty that neither P. bahamense nor P. lunula can be induced to synchrony.
However, G. polyedra would appear to be an ideal choice with which to
examine the correlation of SUSI with reproduction.
T h e data of Fig. 13 show an immediate increase in T S L by a factor of approximately 40 which can be considered as a reversal of photoinhibition. T h e n
at a time corresponding to the natural scotophase, there is a further increase
in T S L by a factor of 100. It is this former reversal of photoinhibition that can
explain the increases and decreases in MSI in situ observed by Backus et al.
(1965) during the course of a solar eclipse. It is apparent that the increases in
T S L per organism shown in Figs. 13 and 14 are under photoperiodic control
and that the arbitrary initiation of a dark interval during photophase, except
for eliminating a relatively small photoinhibition, does not alter the phase of
the expression of TSL. O n the other hand, if photophase is continued past
LI~ there is no observable increase in TSL.
Seliger and McElroy (1968) have demonstrated that concentrations of
natural populations of P. bahamense in Oyster Bay, Jamaica, W. I., can be
assayed by measurement of scotophase M S I and by inference, TSL. T h e present data confirm this for laboratory cultures of G. polyedra and P. lunula as well.
However, since photophase MSI is strongly dependent on ambient light intensity, it is no longer directly related to organism concentration. Thus largescale mapping of P. bahamense concentrations in tropical bays by MSI can be
m a d e only during the night.
Although unfortunately we have not isolated and identified the species responsible, there is at least one bioluminescent plankton in Chesapeake Bay
which does not exhibit a photoperiod-entrained diurnal r h y t h m in stimulable
bioluminescence (Seliger et al., 1961) as well as a second species which does. 4
4 Seliger, H . H . , a n d W . G. Fastie. U n p u b l i s h e d d a t a .
W. H. BIGOLEYET AL. Stimulableand Spontaneous Bioluminescence
I21
K e l l y a n d K a t o n a (1966) h a v e r e p o r t e d a p h o t o i n h i b i t i o n of b i o l u m i n e s c e n c e
w h i c h is r a p i d l y reversed by p l a c i n g the organisms in darkness d u r i n g p h o t o phase. T h e i r t e c h n i q u e for m e a s u r i n g b i o l u m i n e s c e n c e was q u i t e different
f r o m o u r o w n a n d their relative c h a n g e s were several orders of m a g n i t u d e
smaller. I t is therefore difficult to c o m p a r e their results directly with ours.
T h e t e c h n i q u e s a n d types of m e a s u r e m e n t s described in this p a p e r should
p e r m i t a m u c h m o r e q u a n t i t a t i v e assay for the effects of p h o t o i n h i b i t i o n a n d
c h e m i c a l agents o n the expression of the r h y t h m s o f b i o l u m i n e s c e n t potential
t h a n has b e e n available heretofore. I n a d d i t i o n we w o u l d like to e m p h a s i z e
the i m p o r t a n c e of the c o m p a r a t i v e study o f different species, b o t h in l a b o r a t o r y c u l t u r e a n d in n a t u r a l populations, in view o f the interspecies differences
described in this paper.
We would like to thank Mrs. C. Eisner for her invaluable assistance in the maintenance of our laboratory cultures and in the experimental measurements. We also thank Mr. W. Esalas for his assistance in several of the light inhibition measurements.
This work was supported by the Division of Biology and Medicine of the Atomic Energy Comrai~sion, Contract AT (30-1) 2802. The experiments in Jamaica, West Indies were supported under
Atomic Energy Commission Contract AT (30-1) 3480 and the Office of Naval Research, Contract
NR-104-848, Nonr 4010 (16).
Contribution No. (560) of the McCollum-Pratt Institute, The Johns Hopkins University and
Contribution No. (137) of the Chesapeake Bay Institute, The Johns Hopkins University.
This paper is based in part on material presented at the Fifth International Congress on Photobiology, Hanover, New Hampshire in August, 1968.
Receivedfor publication 20 August 1968.
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