Oxygen affects on anemone movement
INTRODUCTION
Anemones within the species Anthopleura elegantissima
(Brandt, 1835) which contain zooxanthellae move from a
shaded area, where the photosynthetic rate of their endo-
symbionts would be reduced, towards a brightly lighted
area. Yet, aposymbiotic individuals distribute themselves
at random when similarly exposed (Buchsbaum 1968).
Pearse (1974) suggested that a chemical either consumed
or produced during photosynthesis of the zooxanthellae may
regulate this behavior. Such photosynthetic products are
liberated to the host anemone (Trench 1971). One chemical
so liberated is oxygen. Paramecium bursaria with zoochlorellae
show positive phototactic responses only under semi-anerobic
conditions (Jennings 1915). Oxygen apparently influences
the phototactic response of Paramecium bursaria and may
affect the movement of Anthopleura elegantissima as well..
I herein report on findings implicating oxygen as the
sole factor regulating the described phototactic response
and as a factor affecting interclonal distribution in
Anthopleura elegantissima.
MATERIALS
Both aposymbiotic and symbiotic (containing only zoo¬
xanthellae) Anthopleura elegantissima were collected from
Oxygen affects on anemone movement
a single intertidal clone (to eliminate genetic differences)
located off Hopkins Marine Station, Pacific Grove, Calif.
In addition, 12 individuals from each of 4 separate clones
were collected for clonal spacing studies.
METHODS AND RESULTS
Behavioral Standards: Phototaxis
The anemones were tested for phototaxis using the
apparatus shown in figure 1. Illumination was supplied
by a single 100 watt light bulb suspended 25 cm. above
the top of the aquarium. A rectangular piece of cardboard
was used to produce a well defined shadow covering half
of the aquarium. The light intensity on the lighted half
of the tank was 250 foot-candles, on the dark side it was
5 foot-candles. Fresh sea water at 13°0-100 ran continuous¬
ly through the tank parallel to the shadow line. Before
each experiment anemones were placed along the intended
shadow line of the aquarium and allowed to settle in
uniform light for 6-12 hours. The positions of the anemones
were recorded at the outset and again after 24 hours of
exposure. All experiments were run in groups of 7 animals
Pigure
each.
The results of tests run with both symbiotic and apo-
symbiotic anemones are shown in table Ia. The difference in
response between the two types of anemones is significant
Oxygen affects on anemone movement
(pe.05, Fishers exact test for independence), with only
symbiotic anemones showing consistent positive responses
to light.
Symbiotic anemones were made to lose their endo¬
symbionts by incubation at 30-32°0 for 2-5 days as described
by Buchsbaum (1968). These anemones reacted like the
naturally aposymbiotic anemones (table Ia).
Each of 10 anemones were subject to two defined spots
of light on opposite sides of their bodies. Eight of the
10 elongated in the direction of the lights as shown in
-equr 2
figure 2. The other 2 moved into the dark.
Oxygen Alteration
Oxygen was bubbled through a column of flowing sea water
which then ran continuously into an aquarium. Another
aquarium was treated similarly using nitrogen aeration.
Winkler analyses (Carritt and Carpenter, 1966) of oxygen
content yeilded: 18.6 ml. 0/1. in the oxygenated tank;
4.5 ml. 02/1. in the nitrogenated tank. Before aeration the
oxygen content of the sea water was 7.2 ml. 00/1. Both
symbiotic and aposymbiotic animals were tested, The test
conditions were otherwise those of the previous light-dark
experiment. The results are summarized in table Ib. Only
symbiotic animals in the oxygen depleted tank showed
consistent and significant phototaxis, aposymbiotic dem¬
onstrated no response to the light (pe.002, Fisher's exact
test for independence).
toble I
fects on anemone movement
Oxygen-taxis
Single anemones were made to settle between 2 barriers
in an aquarium, thereby dividing the tank into 2 separate
chambers (figure 3). The water level was maintained equal
to anemone height to prevent water exchange between the 2
compartments. Oxygen concentration on each side of the
anemone was that of the previous experiments: 18.6 ml.
02/1. and 4.5 ml. 02/1. Experiments were run 24 hours in
the dark. Movement was scored if the anemone moved at
least half its body diamenter towards either side. æfu
Six of the 6 symbiotic anemones and 6 of the 6 apo¬
symbiotic moved towards the oxygen saturated side of the
tank. Both results were significantly different from
random movement (pe.025, chi-squared evaluation).
Induced anemone movement with respect to an oxygen
gradient was also observed during the 30-32°0 incubation
done to purge zooxanthellae. At the end of each period,
surviving anemones (12) were either attached to or clustered
around one of 2 air stones used to aerate the water.
Clonal Distribution: Field Studies
Three aposymbiotic and 3 symbiotic clones of Anthopleura
elegantissima were selected for study in the field. Each
of 12 anemones from the center of each clone was scored
according to its degree of clonal spacing, using the
following scoring system: Zero for anemones touching
clone-mates on all sides, 1 if touching on more than 75%
Oxygen affects on anemone movement
of their body, 2 if more than 50%, 3 if greater than 25%,
4 if not touching but less than 1 cm. from nearest neighbor,
and 5 if greater than 1 cm. from nearest neighbor. The
12 scores from each clone were then summed to quantify
the amount of spacing within each clone.
Results are shown in table II. Aposymbiotic anemones
space themselves significantly further apart than did
symbiotic clones (pe.02, T test for paired comparisons).
fahle
Clonal Distribution: Lab Experiments
Twelve speceimens of symbiotic Anthopleura elegantissima
were collected from each of 4 separate clones. Four test
aquaria were set-up as follows: the first was in the dark
with fresh running sea water; the second was uniformly
illuminated at 250 foot-candles and had running sea water;
the third and fourth tanks were kept in the dark, one with
flowing nitrogenated sea water (4.5 ml.00/1.) and the
other had oxygenated sea water (18.6 ml. 00/1.). To insure
tight clonal settling an inverted 250 ml. beaker was used
to inclose each group as it settled under uniform light
and normal flowing sea water. The settled anemones were
subject to the conditions described above, the beaker was
then removed, and the animals were allowed to move. After
24 hours of exposure the animals were scored using the
system used in the field studies. The 4 clones were run
Cxygen affects on anemone movement
through all of the 4 described conditions, but the order of
exposure to the conditions differjed for each clone.
The results are listed in table III. The clones
showed significantly more spacing in the dark than in the
light (pe.02, T test for paired comparisons). The clones
showed significantly greater spacing under oxygen depleted
conditions compared to oxygen saturated conditions (pe.02.
T test for paired comparisons). There was no significant
difference either between spacing of animals in the light
and those in the oxygen rich-water, or between those in
tble I
the dark and those in the oxygen-poor water.
DISCUSSION
Oxygen is the only photosynthetic product needed to
ensure phototaxis. A symbiotic anemone's phototactic
behavior is stopped by increasing ambient oxygen concen¬
tration. Anemones move away from areas of low oxygen
tension to areas of higher oxygen tension. From these
results it can be inferred that components other than
oxygen are neither necessary nor sufficient in the photo-
tactic response. This reaction must include the ability to
detect an oxygen gradient and to behaviorlly respond to
that gradient. Anemones are apparently able to pick-up
oxygen differences at least as small as those established
by their endosymbionts when illuminated unequally.
Oxygen affects on anemone movement
Aeration of oxygen and nitrogen through sea water
displaces roughly equal amounts of carbon dioxide, thus the
behavior noted cannot likely be attributable to a response
to a carbon dioxide gradient.
Since zoochlorellae release oxygen to their hosts and
very little photosynthetically-fixed carbon compared to
zooxanthellae (Muscatine 1971), demonstration of positive
phototaxis in Anthopleura elegantissima containing only
zoochlorellae would add consideribly to evidence indicating
oxygen as the sole factor influencing phototaxis.
By varying oxygen availability or zooxanthellae
activity, spacing within a clone can be alterred. Under
oxygen stress, clonal anemones move away from water further
depleted of oxygen by adjacent animals. This increased
spacing results in increased oxygen availability. In addition,
free standing individuals have a greater surface area
exposed to the water than do animals in contact with adjacent
clone-mates. In the field it is observed that aposymbiotic
anemones space themselves further apart than do symbiotic
anemones. Aposymbiotic clones likely need this increased
surface area and the more oxygenated water to obtain
oxygen which symbiotic anemones obtain from their endo-
symbionts. Increased spacing is also needed by symbiotic
clones living in areas lower in oxygen content than normal.
Therefore, oxygen appears to be a limiting factor affecting
emone movement
interclonal distribution.
I thank the faculty, staff and students of Hopkins
Marine Station, Stanford University. Special acknowledge-
ment is given to Dr. Robin Burnett for his stimulating
help and advice.
SUMMARY
Anthopleura elegantissima was tested for phototaxis and
possible photosynthetic products (oxygen) responsible for
such behavior. Anemones, symbiotic with zooxanthellae, show
phototactic responses. Phototaxis can be disrupted by
exposing anemones to oxygen contents higher than ambient.
Directional movement is induced by exposing opposite sides
of an anemone to different oxygen concentrations; the
anemone moves towards the side of higher oxygen content.
Therefore oxygen, as a photosynthetic product of zooxanthellae,
is a controlling factor in phototaxis.
Aposymbiotic clones of Anthopleura elegantissima show
significantly more spacing between individuals than symbiotic
clones. Symbiotic clones under oxygen stress show spacing
similar to aposymbiotic clones. There is a direct relation¬
ship between distribution of clonal individuals of Antho-
pleura elegantissima and oxygen availability, either from
the environment or from endosymbionts.
Oxygen affects on anemone movement
LITERATURE CITED
Buchsbaum, V. M., 1968. Behavioral and physiological
responses to light by the sea anemone Anthopleura
elegantissima as related to its algal endosymbionts.
Doctoral dissertation, Stanford University, 13 pp.
Carritt, D. E., and Carpenter, J. H., 1966. Comparison and
evaluation of currently employed modifications of the
Winkler method for determining dissolved oxygen in
seawater. J. Mar. Res., 24 (3): 286.
Jennings, J. A., 1915. Behavior of the Lower Organisms.
Columbia University Press, New York, 366 pp.
Muscatine, L., 1971. Experiments on green algae coexistent
with zooxanthellae in sea anemones. Pac. Sci., 25: 13-21.
Pearse, V. B., 1974. Modification of sea anemone behavior
by symbiotic zooxanthellae: Phototaxis. Biol. Bull.,
147: 630-640.
Trench, R. K., 1971. The physiology and biochemistry of
zooxanthellae symbiotic with marine coelenterates.
Proc. Roy. Soc. London Series B, 177: 237-250.
10
Oxygen affects on anemone movement
2
3
5
light
beams



FIGURE 2
Cxygen afrects on anemone movement
beams









S










14
. .
2


3




12
Cxygen affects on anemone movement
symbiotic
aposymbiotic.
aposymbiotic
(heat-treated)
symbiotic.
high O,
symbiotic
ow O,
aposymbiotic.
high O,
aposymbiotic.
low 0,
Number
in light
14
18
13
TABLE !
Oxygen affects on anemone movement
Number
Number
on midline
in dark

3
5
8
10
6
e
Clone
symblotic A
symbiotie B
symbiotic C
aposymbiotie A
aposymbiotie
aposymbiotie C
Oxygen affects on anemone movement
14
TABLE II
Spacing score
1
3
33
46
43
c
Clone
B
C
Og rich
14
Oxygen affects on anemone movement
15
TABLE III
O2 poor
Light
Dark
33
40
24
23
27
22
22
e
Oxygen affeets on anemone movement
16
LEGENDS FOR FIGURES AND TABLES
Figure 1. Apparutus used in phototaxis experiments.
(a) Horizontal view of aquarium; (b) vertical view.
Figure 2. Phototaxis experiment with spots of light
on opposite sides of anemone, shown at onset (a); and con¬
clusion (b).
Figure 3. Oxygen-taxis apparatus: (a) horizontal view
showing adjustable drains; (b) vertical view, tank sealed
in half by anemone. Oxygen-poor sea water was run into one
side, oxygen-rich into the other.
Table I. Results from phototaxis experiments: (a) with
normal sea water, symbiotic anemones show positive phototaxis;
(b) with oxygen content in the sea water varied, only symbiotic
anemones under low oxygen show positive phototaxis.
Table II. Results taken in field showing greater spacing
of aposymbiotic clones compared to symbiotic. Listings are
sums from scoring of 12 individuals from each clone; high
score indicates a higher degree of spacing.
Cxygen affects on anemone movement
Table III. Lab results of spacing experiments showing
an increase in the dark and under oxygen-poor conditions.
Listings are sums from 12 individual's scores; high score
indicates greater spacing.