Amino Acid Profiles and Density of Symbiotic Zooxanthellae from the Sea Anemone Aiptasia pallida as Potential Bioindicators of Dissolved Inorganic Nitrogen Exposure

Abstract

The waters of South Florida are experiencing nutrient loading from a variety of anthropogenic sources. A bioindicator species which responds to dissolved inorganic nitrogen (DIN) exposure, and can signal locations and times of elevated DIN, may be an alternative to costly, time consuming water quality testing, as well as provide support for changes in anthropogenic practices. Aiptasia pallida, a sea anemone commonly found in South Florida, contains single-celled zooxanthellae which respond to high inorganic nitrogen enhancement (20 and 50 μM NH₄⁺) in the laboratory with an increase in zooxanthellae density and changes in amino acid profiles. The purpose of this study was to determine whether internal free amino acid (IFAA) composition and density of zooxanthellae freshly isolated from A. pallida could be potential bioindicators of inorganic nitrogen exposure at much lower, environmental levels of DIN (0 - 7 μM NH₄⁺), such as those found in the waters of South Florida. Symbiotic zooxanthellae respond to lower levels of DIN (7 μM NH₄⁺) by exhibiting an increase in zooxanthellal total IFAA content for at least four weeks after the removal of the inorganic nitrogen. Moreover, zooxanthellae response to NH₄⁺ seems to be affected by previous DIN exposure, and the nutrient-status of the zooxanthellae. In my preliminary experiment, I exposed the anemones to varying concentrations of ammonium for two weeks. There was no relationship between zooxanthellae densities and increased NH₄⁺ concentration, most likely because the zooxanthellae were nutrient sufficient, not nutrient-limited. An increase in zooxanthellae density is expected in nutrient-limited zooxanthellae. The nutrient-sufficient zooxanthellae of anemones exposed to 20 μM NH₄⁺ had decreased zooxanthellae density, perhaps because of nitrogen toxicity. The symbiotic zooxanthellae were not nutrient-limited during the experiment because the water in which the anemones were maintained before the start of the experiment contained NH₄⁺. This NH₄⁺ in routinely maintained cultures was unanticipated. I added a two-week acclimation period to the second and third phase of the investigation, however, this was not enough time to create nutrient-limited zooxanthellae. After the anemones were acclimated for two weeks in low-nutrient seawater, they ere exposed to 5.38 μM NH₄⁺ for two weeks. Contrary to predictions, the zooxanthellae density decreased significantly in cells per mg protein, during the experimental NH₄⁺ exposure. The Gln:Glu ratio, an indicator of nutrient-status in ooxanthellae, also indicated that the zooxanthellae were nutrient-sufficient throughout the experiments. Zooxanthellae density is apparently influenced by previous inorganic nitrogen exposure and the nutrient-sufficiency of the zooxanthellae. The units used in reporting zooxanthellae density seem to affect the interpretation of the results. Most previous researchers observe an increase in zooxanthellae density when reporting density as cells per cm². Perhaps a decrease in density would be observed, as in my investigation, when density is reported as cells per mg protein. In the third phase of my investigation, I observed zooxanthellal response to the removal of DIN for two weeks after exposure to 5.38 μM NH₄⁺. Again, because zooxanthellae are affected by the previous DIN exposure for at least four weeks, two weeks was not long enough to observe the zooxanthellal response to the removal of the experimental DIN. Anemones collected and examined at four different sites along the Florida Keys also had zooxanthellae densities indicative of nutrient-sufficient zooxanthellae. However, I did not investigate ammonium levels in the field.