California Sea Grant College Program

Many marine sponges possess dense microbial populations, but little is known regarding specific members or interactions of the microbial community within sponges. This study utilized molecular phylogenetic techniques, including quantitative ribosomal RNA (rRNA) hybridization with domain­ specific oligonucleotide probes and phylogenetic analysis of isolated l6S rRNA gene sequences, to assay the diversity of prokaryotes within marine sponges (Xestospongia sp, Cliona celata, and Axinella mexicana). All the sponges harbored diverse prokaryotic assemblages. The majority of rRNA phylotypes were similar to those commonly found in the marine environment. However, the dominance of Prochlorococcus-like rRNA gene clones within Xestospongia sp., and the presence of archaeal rRNA and rDNA within A. mexicana might suggest that these prokaryotes represent sponge-specific microflora.

The association between the marine archaea and A. mexicana was further investigated to determine if the relationship was specific and persistent. rRNA hybridization studies showed that archaea inhabited all specimens of A. mexicana from the Santa Barbara Channel and Monterey, CA, and that the association could be maintained over 4 years in laboratory aquaria. Phylogenetic analysis of the 16S rDNA showed that the archaeal population of a single individual consisted of highly-related strains of an archaeon named Cenarchaeum symbiosum that were affiliated with, but distinct from uncultivated marine planktonic crenarchaeotes. The presence of the strains in natural populations of A. mexicana was investigated using strain-specific 23S rRNA oligonucleotide probes and 16S rRNA signature sequence analysis. Several highly related rRNA phylotypes (>99.2% sequence similarity) were detected in single specimens of A. mexicana. Sequence analysis of the 165 rRNA gene also indicated minor differences in the Monterey and Santa Barbara Channel strains of C. symbiosum. Using in situ hybridization with fluorescently­ labeled 165 rRNA oligonucleotide and 16S/23S ribosomal polynucleotide probes that target marine crenarchaea, the sponge-associated archaea were identified and found to inhabit extracellular regions of the sponge matrix. The results of the above study indicated that the association between A. mexicana and Cenarchaeum symbiosum. is a symbiosis. The association represents the first description of Archaea inhabiting a sponge. and the first specific association between crenarchaea and a metazoan.