Effects of light and thermal variation on symbiotic and aposymbiotic states of the temperate sea anemone, Anthopleura elegantissima  Ellen G. Dow1,2, Allison K. Barner2, Angela Z. Poole2, and Virginia M. Weis2 Department of Biological Sciences1, Florida International University; Department of Integrative Biology2, Oregon State University Abstract Results Mitotic Index Figure 6. Percentage of dividing dinoflagellates in A. elegantissima under light and dark conditions in 12°C, 20°C, and 30°C over 72 hours. Light, temperature, and time are statistically significant factors in the measurement of mitotic indices over 72 hours (ANOVA, linear regression additive model chosen by lowest AIC; light, p=2.130x10-8; temperature, p=2.930x10-5; time, p=2.393x10-7). The temperate aggregating anemone Anthopleura elegantissima can occur in the symbiotic state − with dinoflagellates, when animals reside in habitats with direct light exposure. However, in very low light environments, in caves or on the undersides of rock benches, A. elegantissima may occur in an aposymbiotic state − without dinoflagellates. A. elegantissima is adapted to cool water temperatures in light-deprived regions and desiccating intertidal environments characterized by large temperature variability. A. elegantissima acts as a model system for analyzing physiology, ecology, and molecular biology of temperate cnidarian-dinoflagellate symbioses. To further understand the relationship between symbiotic state in these anemones and the environment, symbiotic and aposymbiotic A. elegantissima were subjected to variations in light and temperature, then sampled over several days to examine algal cell division rate and patterns of gene expression between symbiotic states. Haemocytometer counts were used to calculate mitotic index − percentage of dividing symbionts. QPCR analysis was used to determine relative gene expression. Two genes are associated with the vitamin K cycle: sym32, previously shown to be related to symbiotic state and encode a fasciclin-domain protein and calumenin, an inhibitory protein. Hsp90 and ferritin genes were analyzed as a measure of the effect of heat stress on transcription. Mitotic indices and relative gene expressions demonstrate dynamics of symbiosis relative to light and temperature in A. elegantissima. Relative gene expression in aposymbtiotic and symbiotic A. elegantissima (3-way interaction model) Figure 2. sym32: Symbiosis and light affected gene expression (p=0.000804, p=4.95x10-10). There was a significant difference between 12°C and 20°C (p=0.021386). 48 and 96 hours after initial sampling was significant (p=9.63x10-5, p=4.46x10-10). Figure 7. Dividing dinoflagellate algal symbionts on haemocytometer Figure 3. calumenin: Symbiosis alone did not significantly influence gene expression (p=0.065566). Symbiosis combined with light, temperature, or time at 48 hours had a significant affect (p=0.006649, p=0.001908, p=0.022018). Photos by Ellen Dow Conclusions Symbiosis, light, temperature, and time point interact to influence gene expression in A. elegantissima. Availability of light positively affects expression of sym32 and hsp90 in symbiotic and aposymbiotic anemones. In aposymbiotic anemones exposed to light, calumenin and ferritin (at 12°C) are up-regulated. Symbiosis appears to influence gene expression of sym32, hsp90, and ferritin, particularly in conjunction with heat. However, symbiosis does not appear to affect calumenin expression. Temperature does not seem to be associated with particular trends of gene regulation. Mitotic index decreases over time with increased temperatures and under dark treatments. Figure 1: Aggregating anemone, Anthopleura elegantissima Figure 4. hsp90: Symbiosis and 20°C influenced expression to be differentially significant from the control (p=6.83x10-5). Light and 20°C somewhat influenced gene expression (p=0.03926). Interaction of symbiosis, light, and 20°C affected gene expression (p=0.00162). Differentiation in expression at 48 hours in 20°C (p=0.01506). Aposymbiotic Symbiotic Photos from www.wwu.edu Methods Anthopleura elegantissima collected from Neptune Beach, Oregon Experiments at Hatfield Marine Science Center, Newport, Oregon Tentacle snips sampled at times listed below Mitotic Index 0, 24, 48, 72 hours Cell counts on haemocytometer Relative Gene Expression Real-time quantitative PCR 0, 24, 48, 96 hours Genes of interest: sym32, calumenin, hsp90, ferritin Reference genes: L10, L12, PABP Acknowledgements Set up: Figure 5. ferritin: Symbiosis, light, and temperature did not separately affect gene expression (p=0.34736, p=0.53066, p=0.06034). Symbiosis and 20°C (p=0.01369); light over 48 hours (p=0.01461); light and 20°C at 48 hours (p=9.53x10-5) did produce differences in gene expression. We thank Hatfield Marine Science Center for use of their facilities and sea water flow-through tanks during the experiment. Ellen thanks Dr. Sally Hacker, Dr. Sarah Henkel, Reuben Biel, and Dr. Sarah Close for their assistance in project development and experimental set up at HMSC. We thank Jennifer Mai for assistance in processing samples for qPCR. We also wish to acknowledge the Center for Genome Research and Biocomputing at Oregon State University for sequencing and use of equipment. Key: Replicate number