J. J. Robinson á M. F. Polz A. Fiala-Medioni á C. M. Cavanaugh Physiological and immunological evidence for two distinct C 1 -utilizing pathways in Bathymodiolus puteoserpentis (Bivalvia: Mytilidae), a dual endosymbiotic mussel from the Mid-Atlantic Ridge Received: 3 October 1997 / Accepted: 23 July 1998 Abstract The existence of endosymbiotic sulfur-oxidiz- ing chemoautotrophic and methanotrophic bacteria as- sociating with marine mytilid mussels has previously been inferred by 16S rDNA analysis in Bathymodiolus puteoserpentis Von Cosel et al. 1994, a hydrothermal vent mussel from a site on the Mid-Atlantic Ridge. In mussels collected in June 1993, we found evidence of enzymes diagnostic of two distinct C 1 assimilation pathways in this symbiosis. Assays for the utilization of radiolabelled methane and for immunodetection of methanol dehydrogenase were positive, indicating that oxidation and incorporation of this substrate are oc- curring in this symbiosis. Sul®de or thiosulfate had no detectable stimulatory eect on CO 2 incorporation, and assays for the enzyme ribulose-1,5-bisphosphate car- boxylase/oxygenase (RubisCO), an enzyme diagnostic for the Calvin±Benson cycle, were negative. RubisCO was detected in all samples examined by immunoblot analysis, indicating this enzyme is expressed in the B. puteoserpentis symbiosis. Stable isotope data showed that carbon isotope values were in agreement with pre- viously reported values, and nitrogen isotope values were among the most depleted ever reported for bivalve symbioses. The carbon isotope values do not preclude the utilization of vent-derived methane. These data could be explained by the presence of two metabolically distinct bacterial symbionts or a Type X methanotrophic symbiont. Introduction Symbiotic interactions between autotrophic bacteria and marine invertebrates are an important nutritional adap- tation for organisms living in environments with the appropriate substrates present in relative abundance (reviewed by Fiala-Medioni and Felbeck 1990; Cav- anaugh 1994; Nelson and Fisher 1995). Sulfur-oxidizing chemoautotrophic bacteria (thioautotrophs) are a major symbiont type in species found around hydrothermal vents, tectonic subduction zones, cold seeps, and re- ducing sediments (Nelson and Fisher 1995), while me- thanotrophic (methane-utilizing) bacterial symbionts have been described thus far from cold seeps in the Gulf of Mexico (Childress et al. 1986; Cavanaugh et al. 1987; Fisher et al. 1993), sediments in the Skagerrak (Schmaljohann and FluÈgel 1987), a mud volcano in the Barbados Trench (Vacelet et al. 1995), and one hydro- thermal vent site on the Mid-Atlantic Ridge (MAR) (Cavanaugh et al. 1992). The discovery of thioautotro- phic symbioses at hydrothermal vents (Cavanaugh et al. 1981; Felbeck 1981) precipitated a re-examination of many marine species with questionable nutritional re- quirements and feeding apparatus, and these symbioses have now been described in six phyla (Cavanaugh 1994). To date, methanotrophic symbioses have only been de- scribed in three phyla (Cavanaugh 1993; Vacelet et al. 1995). Evidence has been presented that thioautotrophic and methanotrophic symbionts provide the host with a source of nutrition using carbon, nitrogen, and energy sources which would otherwise be unavailable. Recently, it has been suggested that some bivalve- bacterial symbioses contain both thioautotrophic and methanotrophic bacteria in a single cell type (bacterio- cyte) in the gills (Cavanaugh et al. 1987, 1992; Fisher et al. 1993; Distel et al. 1995). Bacterial morphology provided the ®rst clue that dierent symbiont types may co-exist. For example, in Bathymodiolus puteoserpentis two dis- tinct Gram-negative bacterial morphotypes were ob- served by TEM, one a cell 1.5 to 2.0 lm in diameter with Marine Biology (1998) 132: 625±633 Ó Springer-Verlag 1998 Communicated by J.P. Grassle, New Brunswick J.J. Robinson á M.F. Polz á C.M. Cavanaugh (&) Department of Organismic and Evolutionary Biology, Harvard University, Biological Laboratories, 16 Divinity Ave., Cambridge, Massachusetts 02138, USA A. Fiala-Medioni Observatoire Oceanologique (Laboratoire Arago), F-66650, Banyuls-sur-Mer, France