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.