Trapido-Rosenthal, H.G.*; Zengler, K.; Ritter, J.E.: Multiple Species of Archaea, Cyanobacteria, and Eubacteria Live in Association with the Sponge Chondrilla nucula

Marine sponges have been the subjects of natural products chemists’ investigations for many years, and have been the sources from which novel chemicals with interesting properties have been isolated. Many marine sponges harbor large populations of symbiotic bacteria, an observation which has led to the thought that these bacteria may be the organisms responsible for a significant fraction of the interesting chemicals that have been found in sponge homogenates. Attempts to culture these symbionts prior to further taxonomic and chemical studies have not always been successful. We are investigating the biodiversity of bacterial populations that live in association with sponges using techniques that allow us to bypass the culturing step. The sponge Chondrilla nucula was collected from shallow depths in Harrington Sound, frozen at �70 �C, and then mechanically homogenized in five volumes of sterile, calcium- and magnesium-free artificial seawater (ASW). The homogenates were subjected to multiple rounds of centrifugation on Percoll/ASW gradients (15% v/v). The resulting bands of cells were collected, the cells were lysed, and the 16S ribosomal RNA genes present in the lysate were amplified by means of PCR, using primer sets designed to identify these genes from eubacteria, cyanobacteria, and archaea. The amplification products were cloned, and their sequences determined and compared to 16S sequences in GenBank. The results indicate that 15 species of eubacteria, 7 of cyanobacteria, and 4 of archaea, were living in association with C. nucula. Molecular methods have the potential to enhance our understanding of the nature of sponge-bacteria relationships at both the quantitative and qualitative levels.