FEMS MicrobiologyEcology53 (1988) 159-167
Published by Elsevier
159
FEC 00161
Orientation of the green flagellate, Euglena gracilis,
in a vertical column of water
D o n a t - P . H~ider a n d K a i G r i e b e n o w
Fachbereich Biologie-Botanik, Marbur~ F.R.G.
Received 9 October 1987
Revision received7 December 1987
Accepted 10 December 1987
Key words: Automatic cell counting; Euglena gracilis; Flagellate; Gravitaxis; Photomovement;
Vertical distribution
1. SUMMARY
The orientation of the green flagellate, Euglena
gracilis, in a vertical column immersed in a pond
was studied using automatic cell counting based
on computerized image analysis. When exposed to
solar radiation, the population moved downward
in the column, probably guided by negative phototaxis, and formed a dense layer at the bottom. It is
suggested that this behavior provides an opportunity for the organisms to escape from detrimental
bright light. The downward movement is faster
than the swimming speed of the cells allows and
could be accelerated by a fluid mechanic effect.
The upward movement observed at night may be
due to the precise negative gravitaxis observed in
the organisms. These antagonistic types of behavior allow the organisms to actively search for and
to stay in areas with suitable conditions.
Correspondenceto: D.-P. H~ider,FachbereichBiologie-Botanik,
Karl-von-Frisch-Strasse, D-3550 Marburg, F.R.G.
2. I N T R O D U C T I O N
Like many other motile microorganisms, the
green flagellate, Euglena gracilis, uses external
factors as clues for its orientation and movement
in its environment [1,2]. The cells have been found
to show both step-up and step-down photophobic
responses [3-6] in response to sudden changes in
the fluence rate. In addition, a weak photokinesis
was observed [7]. In laterally impinging light, the
flagellates show either a weak positive phototaxis,
when exposed to fluence rates < 1.5 W • m -2, or a
pronounced negative phototaxis, when exposed to
higher fluence rates [8-12].
Recent results suggest that the cells orient in
light by antagonistic positive and negative phototactic responses [13]; however, w h e n s t u d i e d in a
vertical cuvette, the behavior was found to be
strongly influenced by a pronounced negative
gravitaxis [14], which is also obvious in other
flagellates [15,16]. Thus, the accumulation of
Euglena in a horizontal layer within a body of
water is apparently determined by a very precise
0168-6496/88/$03.50 © 1988 Federation of European MicrobiologicalSocieties
160
negative gravitaxis (plus a weak positive phototaxis at low fluence rates) and an antagonistic
negative phototaxis caused by high fluence rates;
the inversion between upward and downward
movement was found to be at 30 W . m -2 [14].
Phototaxis in this organism is a typical blue
light response [17]. The photoreceptor pigments
are thought to be located in the paraflagellar b o d y
(PFB), a swelling at the emerging flagellum inside
the reservoir [4,18,19]. A flavoprotein is supposed
to be the responsible photoreceptor molecule, as
suggested by the action spectrum measured for
phobic responses and by microspectroscopic analysis [4].
I n the past, the mechanism of orientation has
been explained b y the shading hypothesis [9,20],
according to which the stigma periodically casts a
shadow on the PFB when the cell rotates during
forward locomotion in a lateral b e a m of light.
This shading hypothesis has recently been shown
to be not applicable to the photoorientation in
Euglena [14,21]. Instead, the light direction is detected by the dichroic orientation of the photoreceptor pigments [14] as determined by an automatic tracking system [22].
The aims of this paper are to investigate the
movements of the green flagellate, E. gracilis, in a
b o d y of water and to define the ecological consequences of photobehavior and gravitaxis.
3. M A T E R I A L S A N D M E T H O D S
3.1. Organism and culture
All experiments were carried out with the green
flagellate, E. gracilis, strain Z, which was grown in
100 ml Erlenmeyer flasks filled with 40 ml of a
medium described previously [23-25]. The cells
were grown at 2 3 ° C in a temperature-controlled
room at a constant illuminance of 400 lx from
fluorescent tubes until they reached a concentration of about 2 × 106 cells per ml [21]. These static
cultures were used to inoculate 1.2 1 of the same
medium in 3 1 Erlenmeyer flasks on a planetary
shaker adjusted to 80 rpm.
3.2. Column design
When the cultures reached the concentration
indicated above they were subjected to the irradiation procedure. They were transferred to a plexiglass colunm 1 m in length and with an inner
diameter of 90 m m (Fig. 1). Outlets were installed
starting 50 m m above the bottom at 50 m m intervals which consisted of 40 m m long plexiglass
tubes with 0.5 m m inner diameter pointing towards the center of the column. The outlets were
connected by 1.3 m long silicone tubes with a
peristaltic p u m p (Desaga, model 853418, Heidelberg, F.R.G.), which was designed to draw 18
samples in parallel, which in turn were drained
into multiwell plates (Nunclon, Delta, Denmark)
at predefined regular intervals. The dead volume
in the tubes (approx. 0.25 ml) was discarded before a new sample (approx. 1.5 ml) was drawn.
The column was immersed vertically in a pond in
the Botanical Garden, Marburg, F.R.G. For con-
PUMP
SAMPLE
HOLDER
BE
BE
BE
lie
El
BE
BE
El
lie
|i
|i
dJ
|i
CUVETTE
Fig. 1. Schematic diagram of the column (1000 mm long, 90
mm inner diameter) with 18 outlets connected by silicon tubes
(0.5 mm inner diameter, 1300 mm long) to a peristaltic pump
which handled 18 samples in parallel. At preselected time
intervals 18 samples of about 1.5 ml were drawn simultaneously into multiwell plates.
161
trol purposes the behavior of the population was
studied macroscopically and recorded photographically before and after an exposure and during parallel experiments with solar exposures above
water.
3.3. Cell density determination
The samples were transferred to the laboratory
for counting. Since the large number of samples
made manual counting highly impracticable we
used an automated system for cell density calculation based on image analysis [26]. After thorough
stirring, part of the sample was transferred into a
Thoma chamber and mounted on the stage of an
inverted microscope (ICM 405, Zeiss Oberkochen,
F.R.G.). In order to enhance the contrast for
subsequent image analysis we used dark field illumination. Infrared irradiation was chosen (RG
720, Schott & Gen., Mainz, F.R.G.) in order to
avoid phototactic accumulation or aerotactic responses due to photosynthetic oxygen evolution
by the cells.
The image of the cells was recorded by a CCD
camera (Philips, LDH 0600) at low magnification
( × 6.3). The video image was digitized in a Matrox
digitizer (PIP 512, Matrox, Quebec, Canada) which
occupied one slot in a Picotron IBM compatible
PC microcomputer. After a frame was taken and
digitized the number and sizes of the organisms
visible in the field were analyzed using algorithms
described previously [26]. In order to improve the
statistical significance, several samples were taken
and averaged.
3.4. Light and temperature measurements
The white light fluence rate of the solar irradiation was determined with a digital luxmeter
(Mavolux-digital, Gossen, Erlangen, F.R.G.) and
monitored continuously, measuring the photocurrent from a Si-diode (OSD 60-5T, Centronic,
Croydon, U.K.), which had previously been
calibrated against the luxmeter. In order to convert the fluence rate into the energetic system, the
following conversion factor was measured using a
calibrated thermopile (CA 1, Kipp&Zonen,
Kronberg, F.R.G.), connected to a microvoltmeter
(Model 155, Keithley, U.K.): an illuminance of
100 klx corresponded to an energy fluence rate of
about 1100 W - m -2. The photocurrent was
recorded on a Curken (model 250, Dunbury, CT,
U.S.A.) strip chart recorder. The underwater
fluence rate profile was determined using the Sidiode enclosed in a custom-made, cylindrical
water-tight plexiglass container. Temperature was
measured along the water column after equilibration with the environment.
4. RESULTS
The data shown here were taken on July 12,
1987 and the experiments were repeated during
the following two weeks. Fig. 2 shows the solar
irradiation during this day which was partially
cloudy. The fluence rate reached about 100 klx.
During cloudy intervals the fluence rate dropped
to less than half this value. The transmission of
the body of water next to the inserted column was
determined at various solar fluence rates. A typi-
100
SO
2O
i
|
10-
5-
21-
I
6
I
I
I
I
I
I
I
8
10
12
14
16
18
20
Time IH
Fig. 2. Solar flue,ace rate, measured in klx, recorded during
July 12, 1987.
162
100
A
26
80
i
-
24
-
22
2.,
°~
t~
4O
-
20
20
E
18
0
0
I
200
I
400
I
600
I
800
16
1000
Depth [rnm]
Fig. 3. Optical transmission along the depth of the pond in the Botanical Garden at Marburg, F.R.G. (e) and temperature depth
profile (©) in °C.
cal depth profile is shown in Fig. 3 together with
the temperature profile measured at noon (July 12,
1987). The transmitted fluence rate decreased
steadily to about the 1% level at the bottom of the
pond due to the fairly high turbidity. The temperature dropped in a non-linear fashion (Fig. 3).
Immediately after insertion of the column, samples were taken from the 18 outlets to establish
the initial cell distribution. It should be stressed
that the top outlet was 50 mm below the surface
of the liquid in the column (which was at the same
level as the pond surface) and that the lowest
outlet was 50 mm above the bottom of the colulna.
Even a few seconds after thorough mixing of
the fluid in the column, differences in the cell
densities could be observed between the outlets
(Fig. 4). In order to test whether the population
density deviated significantly from a random distribution, the chi square test was performed on the
data; the results are shown above the density
histograms; they indicate that the initial distribution was not significantly different from a random
distribution. The next samples were drawn at 1 h
intervals. Already 1 h after the onset of the experiment the apparent cell density had decreased
noticeably, which is probably due to the fact that
some of the cells had already moved below the
lowest measuring outlet. After 2 h of solar exposure a pronounced maximum was found in the
cell densities measured at the two bottom outlets.
This maximum decreased during the following 2 h,
indicating that more cells had moved into the
bottom layer below the lowest outlet.
163
X2
19
31
2581
1499
518
I
100
I
200
3OO
4oo
.E
I
600
i
700
I
800
P
900
0
I
m
2
3
4
Exposure time [hJ
Fig. 4. Cell density measured in the samples taken from the 18 outlets of the immersed column at 1 h intervals starting at 10 a.m. on
July 12, 1987. The chi square values for each set of data are indicated above the density histograms.
In order to determine the movements of the
population in more detail, in another experiment
the density distribution was assayed at 10 min
intervals (Fig. 5). Starting with a homogeneous
distribution, the first drastic deviation from this
pattern was observed after 10 rain, when a dense
mass of cells moved downward and passed the
second outlet. This travelling b a n d could be followed in the subsequent measurements as it was
moving downward. These localized high concentrations were due to clouds of organisms aggregating in specific areas as could be observed
macroscopically when the column was placed outside the water (Fig. 6). These clouds were seen to
move rapidly downwards. After an exposure of
several hours most of the cells had reached the
b o t t o m and formed a dense layer (Fig. 7).
When the culture was placed in a shallow container (400 x 400 m m with a depth of 50 ram) the
cells quickly moved to the b o t t o m and aggregated
in dense clusters shortly after exposure to light
(Fig. 8). U p o n microscopical observation the
flagellates were found to be immotile and a number of cells aggregated in close physical contact.
Only in areas shaded by the side walls could
clouds of motile ceUs be observed.
During nighttime an upward movement of the
organisms was observed in the column (Fig. 9).
However, this m o v e m e n t was much slower than
the downward movement; after 2 h a dense population started to build up at the b o t t o m outlet and
an obvious upward m o v e m e n t was seen only after
4 h. This distribution did not change considerably
during the following hours (data not shown), indi-
164
X2
36
56
17
II
100
r
400
i
I
r
I
I
600
10
p i
!
200
3OO
23
/
700
I
800
|
I
900
0
10
20
I
I
ii
30
40
Expooure time [min]
Fig. 5. Cell density measured in the samples taken from the 18 outlets of the immersed column at 10 min intervals. The chi square
values for each set of data are indicated above the density histograms.
Fig. 6. Clouds of organisms forming in the column during downward movement.
Fig. 7. Dense aggregation of cells at the bottom of the column after 4 h of solar irradiation from above.
Fig. 8. Clusters of
Euglena at the bottom of a flat container. Only in shaded areas are clouds of motile organisms visible.
165
X2 310
465
1890
2121
51
100
I
200
300
1
400
a~"
I
500
I
600
1
700
900
0
llll
I
L L
1
2
3
k
4
Exposure time [h]
Fig. 9. Cell density measured in the samples taken from the 18 outlets of the immersed column at 1 h intervals starting at 7 p.m. on
July 12, 1987. The chi square values for each set of data are indicated above the density histograms.
cating that the upward movement is far slower
than the downward movement.
5. D I S C U S S I O N
The net movement of the green flagellate, E.
in a vertical column of water in a pond
was found to be governed by photo-orientation in
bright daylight and gravitaxis at night. The escape
reaction due to negative phototaxis could be an
efficient mechanism to avoid potentially harmful
white light fluence rates which have been found to
photobleach the cells even at levels lower than
bright sunlight [24]. Simultaneously, this behavior
also partially alleviates the potentially harmful
effects of solar UV-B irradiation which impairs
gracilis,
both motility and photoorientation even at current
fluence rates [13,27].
When the movements of the population were
studied macroscopically in the column taken out
of the pond, the cells were found to form dense
clouds which moved down the column at speeds
higher than could be explained by the cells' swimming speed which in other flagellates has been
attributed to the fluid mechanical properties of
dense suspensions [15,16]. The upward movement
during the night was slower than the downward
movement and seems to be based solely on negative gravitaxis and active cell swimming.
An additional mechanism to obtain some protection from bright white and UV-B irradiation is
by mutual shading caused by the observed dense
aggregation of cells in clusters at the b o t t o m of a
166
shallow layer. The patterns were subject to rapid
variation when the fluence rates changed, due to a
partial cloud cover.
The results reported here are in contrast to a
study on the diurnal movements of various
planktonic organisms in a fish pond in India [28]
where an unidentified Euglena species was found
to move to the surface at daytime (maximum at
noon) and to swim to the bottom at night. Whether
this discrepancy is due to differences in the species
or the physical and chemical parameters of the
body of water cannot be said.
In summary, the orientation of the green flagellate, E. gracilis, in a vertical column immersed in
a pond is governed by the antagonistic effects of
photoorientation and gravitaxis which could provide an effective means to bring the population
into areas of optimal light conditions for growth
and survival and to avoid potentially harmful
white light and UV-B irradiation.
ACKNOWLEDGEMENTS
This work was supported by the Deutsche Forschungsgemeinschaft (Ha 985/5-6) and the
Bundesminister fiir Forschung und Technologie
(KBF 57). The authors gratefully acknowledge the
skillful technical assistance of N. Gorny, M. Hermans, A. H~tberlein, C. Link, E. Reinecke and M.
Rudyk.
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