Bleaching, Recovery, and Growth of Glass Anemone, Aiptasia pallida, as a Result of a Heat Stress. Marina Arruda, Keatyn Bucknam, and Esmeralda Montes Department of Biological Sciences Saddleback College Mission Viejo, CA 92692 Photosynthetic dinoflagellates, Symbiodinium spp. not only have a symbiotic relationship with glass anemones, but also provide energy and food. Both glass anemones and photosynthetic dinoflagellate require precise environmental parameters to survive, therefore unfavorable conditions would cause anemones to stress and undergo a process called bleaching by releasing their endosymbionts. Therefor it was predicted that A. pallida that had previously recovered from a bleaching event would bleach and recover more at a different rate than anemones that had not experienced any stresses. Two 2.5 gallon tanks each containing varying sizes of anemones were divided to house two different groups: those growing in ideal environmental conditions, and those having recovered from a previous bleaching event. The temperature in the experimental tank was gradually increased to 32°C for 48 hours, and then reduced to normal temperature. The color value change for bleaching was then obtained by subtracting the initial value from the maximum color value, indicating a significance of bleaching color change (ANOVA, p=0.0079), that occurred between the Experimental Normal group, Control Normal and Control Recovered groups (Post Hoc-Bonferroni Correction, p<0.05). The Experimental Normal group revealed a color change during recovery that was statistically different than that of the other groups, including the experimental group, having previously recovered from a stress and being exposed to the same condition, showing that there was a significant difference between recovered and normal anemone bleaching and recovery as a result of a heat stress. Introduction Glass anemones (Aiptasia pallida) have a symbiotic relationship with a photosynthetic dinoflagellate, Symbiodinium spp. (Muller-Parker & Davy, 2001 and Weis, 2008), which provides additional energy and food to the anemones and gives the anemones their color. A. pallida and Symbiodinium require specific environmental parameters to survive which include water temperature between 24.0-26.0°C, specific gravity of 1.024-1.027, pH 8.09.0, levels of ammonia between 0.01.0ppm, nitrate levels of 0.0-40.0ppm, nitrite levels of 0.0-5.0ppm, and phosphate levels of 0.0-5.0ppm; unfavorable conditions will cause the anemones to stress and release their endosymbionts (a process called bleaching). If adequate food is not available, they perish quickly without the nutrients necessary to survive (Wissmann, 2003). Multiple stresses have been shown to cause bleaching in A. pallida. For instance, a study by Arruda et al. (2013) demonstrated that anemones placed in complete darkness bleach because their photosynthetic endosymbionts are unable to undergo metabolism without the presence of light. Another avenue of bleaching was demonstrated in a study by Dunn et al. (2007), which showed that a temperature increase between 33-34°C over a period of 30 hours caused A. pallida anemones to bleach. This method of bleaching is comparable to coral bleaching induced by a heat stress, since the most pressing cause of coral bleaching is due to climate change (Hughes, et al.). The present project will study the color change due to bleaching and recovery of A. pallida after they have been exposed to a heat stress (increase in temperature) for a short period of time. The purpose of this experiment is to compare the bleaching and recovery of anemones that have bleached and recovered prior to the experiment (recovered) and anemones that have grown in ideal conditions (normal) in order to determine if there is a correlation between recovery and previous bleaching events. It is proposed that there will be a significant difference between recovered anemone and normal anemone bleaching and recovery. This could provide further insight into the loss of coral reef habitats due to coral bleaching. Materials and Methods A total of 124 Aiptasia Pallida anemones maintained from a previous study (Arruda et al., 2013) previously obtained from Berghia Breeder (Laguna Niguel) were monitored over a period of 7.5 weeks. The anemones were separated into four groups and kept in two 2.5-gallon glass tanks (Petco, Aliso Viejo). Groups included anemones recovered from a previous bleaching event in the experimental tank (experimental recovered), anemones that have grown in ideal conditions in the experimental tank (experimental normal), anemones recovered from a previous bleaching event in the control tank (control recovered), and anemones that have grown in ideal conditions in the control tank (control normal). One 2.5-gallon tank, placed on top of an egg crate (Home Depot, Laguna Niguel) within a 30-gallon glass tank obtained from a Saddleback College student (Danielle Breski) housed the experimental group of anemones. The other 2.5-gallon tank housed the control group and was separate from the 30gallon tank. Each 2.5-gallon tank housed six large (>2.5cm), six medium (12.5cm), and fifty small (<1cm) anemones. A porous ceramic cube (Reefapalloza, Anaheim) was placed in the corner of each 2.5-gallon tank. Tank dividers were constructed from clear plastic canvas (Michael’s, Aliso Viejo) and plastic slide locks cut to fit between each individual 2.5-gallon tank to separate the recovered from the normal anemones in each tank. These were soaked in reverse osmosis deionized water, RODI, (Sand Bar Pet Shop, Mission Viejo) prior to installation. The dividers were placed in the middle of each tank and did not restrict the flow of food, heat or water throughout the tanks, but prevented recovered and normal anemones from mixing. On the bottom each tank, 1×2 cm ceramic tiles (Home Depot, Laguna Niguel) were placed on top of 10×5 cm ceramic tiles (Home Depot, Laguna Niguel) to allow easier anemone removal for diameter measurements and pictures. The 2.5 gallon tanks were filled approximately 1.5 cm from the top with saltwater (Sand Bar Pet Shop, Mission Viejo), and the 30 gallon tank was filled up to 2.5 cm from the smaller tanks with tap water (no water circulation between the 30 gallon and 2.5 gallon tanks). All the materials that were placed into the tank or came in contact with the tanks were rinsed with RODI prior to installment. In the corner of each 2.5-gallon tank, a Hydor centrifugal water pump was installed (Amazon, LLC). An aquarium 2-way gang valve and a fusion air pump were installed in order to direct airflow within the tanks (Amazon, LLC). Both tanks received light via a light hood 3.48cm away (Danielle Breski). An Aquatop GH Series 150 watt Submersible Heater (Amazon, LLC) was placed inside the 30-gallon tank, while a Hydor 25-watt submersible aquarium heater (Amazon, LLC) was placed into the Control 2.5-gallon so that its temperature would remain independent of that within the experimental tank. Water parameters within each 2.5-gallon tank were measured and tested in the afternoon or early evening each day. In order to measure the salinity of the controlled and experimental tanks, a salinity refractometer (Ade Advanced Optics, Amazon, LLC) was used and calibrated with RODI water. API tests were used to measure ammonia, nitrate, nitrite and phosphate levels (Amazon, LLC) by collecting samples from each tank with their corresponding plastic graduated pipettes (Lake Charles Manufacturing, Amazon) and placing the water samples into separate glass vials or test tubes (Amazon, NPS), then conducting the appropriate tests. The water from each tank was also tested for pH using a high range pH test kit (The Paws, Amazon, LLC). All water parameters, except temperature in the experimental tank, remained consistent at ideal levels in both aquariums (Temperature 24.0-26.0 °C, pH 8-9, Ammonia 0-1 ppm, Nitrate 0-40 ppm, Nitrite 0-5 ppm, Phosphate 0-5 ppm, Specific Gravity 4.1-4.8 %, Alkalinity 180-300 ppm). Both heaters were initially set to maintain a water temperature of 24.026.0°C. Preliminary photographs and diameter measurements were taken for each anemone before the experiment began. Once the experiment began, the control aquarium was maintained at the ideal temperature (24.0-26.0°C), while the experimental aquarium was stressed to a higher temperature of 32.0-33.0°C for a period of 48 consecutive hours and then returned to ideal temperature for the rest of the experiment. Temperature within each tank was measured using coral life digital thermometer (Sand Bar Pet Shop, Mission Viejo). Before starting the photograph and diameter measurement procedures, the specimens were fed with 0.05g of Cyclop-Eeze freeze-dried Mysis Shrimp (Sand Bar Pet Shop, Mission Viejo). Once the anemones finished eating, they were transferred on their tiles into a glass 10×13 inch rectangular dish, owned by Saddleback College student Marina Arruda, that was filled with water that had previously been siphoned out from their own tanks with aquarium airlines (Amazon, LLC). Anemones were transferred and photographed one tank at a time, and with recovered and normal groups kept separate. After allowing the anemones to acclimate, photographs were taken with a Panasonic DMC-TS20 camera (BestBuy, Laguna Niguel) at approximately 6 cm way from each anemone and each diameter was measured. Once the measurements were recorded, the anemones were placed back into their appropriate tank and the water from the dish was siphoned back into that tank through a filter (to remove excess algae). This process was repeated for the second tank. This procedure was done before the heat stress, 24 hours after the experimental tank was brought back to ideal temperature, every 7 days for two weeks, and every 14 days for the remaining four weeks. In order to analyze the photographs, the histogram feature of Photoshop Elements 6.0 was used. For large and medium anemones, three 0.5x0.5mm areas of separate tentacles were analyzed at 100% zoom and averaged to get a color value for each anemone. For small anemones, one 0.5x0.5mm area was used, which encompassed most of the anemone tentacles. Small anemone color values were averaged for each tank. Larger color values indicated a lighter color (greater amount of bleaching). Data was placed into MS Excel (Microsoft, Redmond, WA) and comprised into a line graph of color value as a function of date measured. Bleaching color value change was obtained by subtracting the initial color value from the maximum color value for each group and groups were analyzed against each other using an ANOVA with Post Hoc test (Bonferroni correction). Recovery color value change was obtained by subtracting the final color value from the maximum color value for each group and analyzed accordingly. Diameter measurements were graphed in a line graph as the average diameter of the small anemones in each tank as a function of day measured to show a general growth trend. Results 120 Color Value 100 80 60 40 20 0 31-Jan 10-Feb 20-Feb 2-Mar 12-Mar 22-Mar 1-Apr 11-Apr Date Measured ER EN CR CN * * * Figure 1. Bleaching and recovery rates for Experimental Recovered (ER, N=7), Experimental Normal (EN, N=7), Control Recovered (CR, N=7), and Control Normal (CN, N=7). There is a significance between bleaching color change (ANOVA, p=0.0079). Represents groups that exhibit a significant difference of bleaching color change against the Experimental Normal group (Post Hoc- Bonferroni Correction, p<0.05). There is a significant difference between recovery color changes (ANOVA, p=0.0001). * Represents groups that exhibit a significant difference of recovery color change against the Experimental Normal group (Post Hoc- Bonferroni Correction, p<0.05). Average Diameter (cm) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Control Normal Control Recovered Experimental Normal Experimental Recovered 0 2 4 Day Measured 6 8 Figure 2. Average diameter of small anemones as a function of day measured showing a general growth rate for Control Normal, Control Recovered, Experimental Normal, and Experimental Recovered groups. The four groups of anemones were analyzed to determine their color value change for bleaching and recovery. Each group had three large, three medium, and twenty-five small anemones present. Figure 1 indicates the average of color values (three large, three medium, and one small were added together and averaged; the small color values were averaged, giving one value) for each day they were measured. The color value change for bleaching was obtained by subtracting the initial value from the maximum color value (the lightest color the anemones reached, therefore their maximum bleach color). There was a significant color difference (ANOVA, p=0.0079) between the Experimental Normal group (color lightened) and the Control Normal and the Control Recovered groups, which showed no significant change (Post HocBonferroni Correction, p<0.05). The color value change for recovery was obtained from subtracting the final value from the maximum value. There was a significant difference of recovery color change (ANOVA, p=0.0001) between the Experimental Normal group and the Experimental Recovered, Control Normal, and Control Recovered groups (Post Hoc- Bonferroni Correction, p<0.5). Figure 2 represents the general growth trends for each group of anemones. During the initial four weeks, the control groups indicate a higher growth margin than the experimental groups. Between day one and two of measurements (before and after the heat treatment), some of the anemones in the experimental tank died, indicated by the drop in average size. After week four, the control groups exhibit a drop in size because new polyps started to grow, reducing the average diameter values. In the experimental groups, there were no new polyps, so the average diameter values continued to increase. Water parameters remained ideal for each tank during the experiment. The control tank temperature ranged between 24.0 and 25.6 °C. During the temperature stress, the experimental tank temperature reached 32.0 °C and was brought back down to between 24.0 and 26.0 °C to allow for recovery. The control tank parameters were: alkalinity 180-300ppm, salinity 4.2-4.5 %, pH 88.4, nitrate 0-20ppm, nitrite 0-0.45ppm, ammonia 0-0.6ppm, and phosphate 0ppm. The experimental tank parameters were: alkalinity 180-300ppm, salinity 4.1-4.8 %, pH 8-8.3, nitrate 0-20ppm, nitrite 0-0.45ppm, ammonia 0-0.6ppm, and phosphate 0ppm. Discussion The higher color value indicates that the anemones became lighter in color (the higher the value, the closer the color is to white). The color of A. pallida is derived from their endosymbiotic dinoflagellates Symbiodinium, so the lightening indicates that some of these dinoflagellates have been released into the water column or have perished (the anemones have bleached). The experimental normal group showed a color change during recovery that was statistically different than all of the other groups, including the experimental group that had previously recovered from a stress and was exposed to the same conditions. As a result, the null hypothesis can be rejected, and the statistical analysis shows that there is a significant difference between recovered and normal anemone bleaching and recovery as a result of a heat stress. The bleaching of normal anemones in this experiment supports past studies concerning the bleaching of A. pallida as a result of a heat treatment at 32-33°C for a period of 24-48 hours (Ferrier-Pages et al., 2007). The results of this experiment suggest that anemones that have previously recovered from a bleaching event may be more resilient to a future bleaching event, since the experimental recovered anemones did not show a significant change in color when compared to the control groups. This warrants further study on the subject, which could be useful to testing the Adaptive Bleaching Hypothesis (Kinzie, Robert A, et al., 2001). Figure 1 indicates a slight fluctuation in the color values for the control anemone groups (recovered and normal) and the experimental recovered. This fluctuation appears to be a natural phenomenon, as it was observed in all anemones (except the experimental normal group, which showed significant bleaching and recovery due to the heat stress). It also did not have any correlation with water parameters, which were kept within the ideal ranges for the species. The experimental recovered anemones did appear to have some color values within the experimental period that were higher than those in the control groups, but this is understandable since the experimental group was exposed to the heat stress. It is important to note however, that though this difference is visible in the figure, it was not significant. During this experiment the diameters of the anemones were measured and recorded in order to determine the general trend in growth of the smaller anemones in each group during the experimental period. As seen in Figure 2, the control group diameters steadily increased during the experimental period until new polyps began to grow, which decreased the average diameter values for the tank. The experimental groups showed a decrease in diameter after the heat treatment, then an increase as recovery took place. In the previous study involving these anemones (Arruda et al., 2013) growth appeared to be reduced in anemones that bleached. This suggests that the initial observed decrease in diameter is a result of the stress on the anemones, since more resources must be devoted to capturing food if some of the symbiotic dinoflagellates are absent due to bleaching. The results of this project may shed light on the effects of bleaching in coral reefs due to a temperature stress brought on by climate change. The cause of death from a bleaching event to the anemone or coral is usually due to the decrease in nutrient intake because of the loss of endosymbionts, not because of the actual stress (Wissmann, 2003). During this study, anemones were consistently fed with high nutrientcontent food, allowing them to survive and recover from the loss of those extra nutrients derived form their endosymbionts. Corals and anemones possess a capability referred to as acclimization, which allows the organism to tolerate changing conditions that lie within certain parameters dependent on species (Gates and Edmunds, 1999). The temperature increase therefore causes bleaching in A. pallida, but the loss of their endosymbiotic dinoflagellates (and therefore their source of many essential nutrients) causes death. Acknowledgments We would like to thank Professor Teh for his assistance and guidance with this study. We would also like to thank Danielle Breski and Patrick O’Hara for supplying some of our materials. Authorship was determined alphabetically by last name and does not reflect individual contribution to the study. Literature Cited Arruda, M., Breski, D., Moghadasi, M., Fastuca, J. (2013). The Effect of Darkness on Bleaching in Glass Anemones, Aiptasia pallida. Saddleback Journal of Biology. Dunn, S.R., Schnitzler, C.E., and Weis, V.M. (2007). Apoptosis and Autophagy as Mechanisms of Dinoflagellate Symbiont Release during Cnidarian Bleaching: Every Which Way You Lose. Molecular and Cellular Proteomics, 30793085. Ferrier-Pages, C., Richard, C., Forcioli, D., Allemand, D., Pichon, M., Malcolm Schick, J (2007). Effects of Temperature and UV Radiation Increases on the Photosynthetic Efficiency in Four Scleractinian Coral Species. Biological Bulletin, Vol. 213, No. 1, 76-87. 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