The isolation of a class of bioactive aromatic alkaloid compounds known as pyridoacridines from members of four phyla (Porifera, Chordata-Subphylum Tunicata, Mollusca and Cnidaria) caused some to speculate that they were produced by associated symbionts. We tested this hypothesis by localizing specific metabolites in cells using a combination of visualization methods, including laser-scanning confocal, epifluorescence, and transmission electron microscopy, as well as cell-separation techniques, and chemical analysis. This study demonstrates that large quantities of the pyridoacridine alkaloid dercitamide (=Kuanoniamine C) are localized exclusively in bacteria-free sponge cells in the marine sponge Oceanapia sagittaria (Sollas), and are probably not produced by intracellular symbiotic organisms. We hypothesize that it is unlikely that the pyridoacridines are produced by extracellular bacteria and then transferred to specific sponge cells. The localization of dercitamide in significant concentrations in specific cells throughout the sponge suggests important biological and ecological functions, such as chemical defense against predators and possibly microbial pathogens. If pyridoacridines are produced by the host organism in other phyla, this may be a case of convergent evolution of an efficient and useful biosynthetic pathway.
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Acknowledgements We thank the Coral Reef Research Foundation for assistance with collection of the sponge and use of their research facilities. We are also grateful to the Republic of Palau for research and collecting permits to conduct our studies. M.K. Harper provided valuable assistance with the identification of the sponge and advice about the cell studies. We also thank G. Mitchell for the generous use of the fluorometer and L. Washington, N. Holland, and C. Graham for assistance with preparation and analysis of the electron-microscope samples. We are grateful to three anonymous reviewers who provided useful comments to improve this manuscript. This research was funded by NSF grant CHE-9527064 and NIH grant CA49084 to D.J. Faulkner. Some of the work included here was conducted at the National Center for Microscopy and Imaging Research, which is supported by NIH grant RR04050 to M.H. Ellisman.