The giant hydrothermal vent tubeworm Riftia pachyptila was first found to be symbiotic with intracellular carbon-fixing sulfide-oxidizing bacteria in 1981 (Cavanaugh et al. 1981; Felbeck, 1981). R. pachyptila supplies its symbionts with inorganic carbon, oxygen, hydrogen sulfide and nitrate, which are taken up from the environment across the plume (Arp et al. 1985; Felbeck and Childress, 1988; Childress and Fisher, 1992; Lee and Childress, 1994). These substances are transported in the vascular blood to the bacteria located in an organ known as the trophosome (Jones, 1981). The trophosome is highly vascularized and surrounded by non- circulating coelomic fluid, which is in equilibrium with the circulating vascular blood for smaller molecules such as CO 2 (Childress et al. 1984, 1991). These worms have two extracellular hemoglobins in the blood that bind and transport both oxygen and hydrogen sulfide to the symbionts (Arp et al. 1985, 1987). Inorganic carbon accumulation and transport to the bacteria, however, apparently takes place without significant binding or buffering by blood proteins (Childress et al. 1993; Kochevar et al. 1993; Toulmond et al. 1994). 883 The Journal of Experimental Biology 200, 883–896 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JEB0422 Riftia pachyptila is the most conspicuous organism living at deep sea hydrothermal vents along the East Pacific Rise. To support its large size and high growth rates, this invertebrate relies exclusively upon internal chemosynthetic bacterial symbionts. The animal must supply inorganic carbon at high rates to the bacteria, which are far removed from the external medium. We found substantial differences in body fluid total inorganic carbon (∑CO 2 ) both within and between vent sites when comparing freshly captured worms from a variety of places. However, the primary influence on body fluid ∑CO 2 was the chemical characteristics of the site from which the worms were collected. Studies on tubeworms, both freshly captured and maintained in captivity, demonstrate that the acquisition of inorganic carbon is apparently limited by the availability of CO 2 , as opposed to bicarbonate, and thus appears to be accomplished via diffusion of CO 2 into the plume, rather than by mediated transport of bicarbonate. The greatly elevated P CO2 measured at the vent sites (up to 12.6 kPa around the tubeworms), which is a result of low environmental pH (as low as 5.6 around the tubeworms), and elevated ∑CO 2 (as high as 7.1 mmol l -1 around the tubes) speeds this diffusion. Moreover, despite large and variable amounts of internal ∑CO 2 , these worms maintain their extracellular fluid pH stable, and alkaline, in comparison with the environment. The maintenance of this alkaline pH acts to concentrate inorganic carbon into extracellular fluids. Exposure to N-ethylmaleimide, a non- specific H + -ATPase inhibitor, appeared to stop this process, resulting in a decline in extracellular pH and ∑CO 2 . We hypothesize that the worms maintain their extracellular pH by active proton-equivalent ion transport via high concentrations of H + -ATPases. Thus, Riftia pachyptila is able to support its symbionts’ large demand for inorganic carbon owing to the elevated P CO2 in the vent environment and because of its ability to control its extracellular pH in the presence of large inward CO 2 fluxes. Key words: tubeworm, Riftia pachyptila, inorganic carbon, hydrothermal vent, ion transport, carbon fixation, symbiosis, pH regulation, N-ethymaleimide. Summary Introduction INORGANIC CARBON ACQUISITION BY THE HYDROTHERMAL VENT TUBEWORM RIFTIA PACHYPTILA DEPENDS UPON HIGH EXTERNAL P CO 2 AND UPON PROTON-EQUIVALENT ION TRANSPORT BY THE WORM SHANA K. GOFFREDI*, JAMES J. CHILDRESS, NICOLE T. DESAULNIERS, RAYMOND W. LEE†, FRANCOIS H. LALLIER‡ AND DOUG HAMMOND§ Oceanic Biology Group, Marine Science Institute and Department of Biological Sciences, University of California, Santa Barbara, CA 93106, USA Accepted 12 December 1996 *e-mail: goffredi@lifesci.ucsb.edu. †Present address: Department of Biology, Biolabs 16, Divinity Avenue, Harvard University, Cambridge, MA 02138, USA. ‡Present address: Laboratoire d’Ecophysiologie, Station Biologique, CNRS, BP 74, 29682 Roscoff Cedex, France. §Present address: Department of Geological Sciences, University of Southern California, University Park, Los Angeles, CA 90089, USA.