Development of an Optimized Protocol for the Cytological Evaluation of Glass Anemones (Aiptasia pulchella)
Abstract
Coral reefs are known as some of the most biodiverse and complex ecosystems in the world, containing an estimated 25% of the world’s marine fish species in less than 0.1% of the ocean’s floor,1 with significant ecological, economic, and culture significance. As of 2008, 19% of coral reefs had died with continually increasing rates of decline.2 Research on stony corals is hampered by the inability to remove coral cells from their calcium carbonate skeleton and difficulty maintaining them in a laboratory environment. A closely related symbiotic Anthozoan, the glass anemone Aiptasia provides an ideal model for studying coral as they lack a skeleton, are easily maintained in a laboratory and are capable of both asexual and sexual reproduction.3 To dissociate the organism for cytology, Aiptasia were first relaxed in a bath of 2.5% MgCl2 in artificial sea water (ASW) for euthanasia then macerated with sharp scissors. Aiptasia fragments were then placed in one of three maceration solutions: 1:1:13 glycerin:glacial acetic acid:3% NaCl in DI H2O (GAS), 1:13 glycerin:3% NaCl in DI H2O (GS), or 1:13 glacial acetic acid:3% NaCl in DI H2O (AS).4,5 After dissociation, the cellular monolayer was stained with modified Wright-Giemsa and evaluated under compound light microscopy. While fine cellular detail was best preserved in GS, cellular dissociation was most complete in GAS and AS indicating that acetic acid may play a role in dissociation of Aiptasia tissue. Identifiable cellular components included epithelial cells, secretory cells, zooxanthellae, bacteria, spirocysts, and several stages of nematocysts including microbasic p-mastigophores, B-mastigophores, and holotrichous isorhiza. Here we present an optimized protocol that will be useful in obtaining high quality diagnostic results in the cytologic evaluation of Aiptasia.
Acknowledgements
The authors wish to thank the staff of the Kewalo Marine Laboratory for their support of the project.
* Presenting author
+ Student presenter
Literature Cited
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