Phylogenetic Analysis of Bacterial Endosymbionts in Deep-sea Clams from the West Pacific
Xiufen SUI*, Chiaki KATO, and Koki HORIKOSHI
The DEEPSTAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka, 237 Japan
Several chemoautotrophic animal communities have been found at deep-sea hydrothermal vent and cold seep environments. Such animals which are mostly invertebrates often lack digestive organs but harbor chemoautotrophic endosymbiont in a specialized tissue. In early studies of these symbionts, people believed that only one kind of chemoautotorophic bacteria could exist in one species of the host animal, and that they were sulfur-oxidizing (thioautotorophic) bacteria (1) or methanotrophic bacteria (2). But recently, two separate intracellular symbionts were identified from a single host species, a Mid-Atlantic-Ridge hydrothermal vent mussel (family Mytilidae), and they were affiliated with thioautotorophic and methanotrophic symbionts (3). These chemoautotorophic symbionts are closely related to bacteria of the Proteobacteria γ-subgroup (1-3). However, Proteobacteriaε-subgroup bacteria are also associated with the chemoautotorophic animals as epibionts and symbionts (4, 5), we are interested in how many kinds of such symbiotic microorganisms can be found in the same host species.
The aim of this study is to determine the identification of endosymbionts from the deep-sea clams of the genus Calyptogena, and the nature of the host-symbiont relationships. We extracted bacterial DNA from homogenized gill tissues from Calyptogena specimens collected from deep-sea cold seeps (Sagami-Bay, Nankai Trough, and Japan Trench land side slope) and hydrothermal vents (Okinawa Trough). Extracted DNA was amplified by PCR using specific primers for 16S rDNA (6), then a 16S rDNA library was constructed in Escherichia coli. Several different clones were selected by RFLP-PAGE analysis using the endonucleases, RsaI and MspI, and their nucleotide sequences were determined using specific primers reported by Kato et al. (6). Preliminary results suggest that different kinds of 16S rDNA sequences are present in Calyptogena gill tissue, which might represent different endosymbionts.
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