Ultratome V, stained with uranyl acetate and lead citrate, and 

 examined with a Philips 300 transmission electron microscope. 



NaH14C03 Incubations 



To test for substrate stimulation of carbon fixation by 

 tissues, small pieces (0.2-0.6 g wet weight) of gill or 

 preparations of vestimentiferan trophosome were incubated with a 

 variety of substrates in 10 ml glass syringes, at 7.5xC, with 

 NaH CO3 ( ICN Radiochemicals, Irvine, Ca., specific activity = 54 

 mCi/m mol ) . Gills were dissected from the mussels and cut into 

 small pieces without damaging individual filaments. The pieces 

 of gill were rinsed in 0.45 fm membrane filtered seawater (MFSW) 

 and then preincubated for one hour in MFSW with the substrate to 

 be tested. Following preincubation, the gill pieces were placed 

 in 10 ml glass syringes containing fresh MFSW and the desired 

 substrate (and/or inhibitor). After an additional 10r-20 minutes 

 the incubations were initiated by the addition of NaH CO3 

 (200fCi/ml) to a final activij^ of 0.5-2.0 fCi/ml. At the end of 

 the incubation period the NaH CO3 fixation was stopped by 

 removing the gill pieces from the syringes, quickly blotting them 

 dry, and freezing the pieces in cryovials in liquid nitrogen. 

 Viability of the gill tissue throughout the incubation period was 

 confirmed by visual observation (under a dissecting microscope) 

 of ciliary currents on the surface of other pieces of gill tissue 

 removed from the same animals. 



Trophosome was dissected free from the anterior portion of 

 the trunk of the vestimentif erans and tissue containing bacteria 

 was separated from the major blood vessels and gonads also 

 present in this organ. A portion of the tissue (0.49-0.71 g) was 

 quickly weighed on a motion compensated shipboard balance system 

 (Childress and Mickel 1980) and then submerged in 20 ml of 

 deoxygenated (nitrogen purged), pH 7.5 vestimentiferan saline 

 solution . The tissue was gently homogenized for 5-10 seconds in 

 a chilled, loose fitting Dounce type ground glass tissue 

 homogenizer, to rupture the bacteriocytes and disperse the 

 symbionts. A portion of this homogenate was fixed in buffered 

 glutaraldehyde and integrity of the bacteria was later 

 confirmed microscopically in the laboratory. To commence the 

 incubations, NaH CO3 was added to the trophosome preparation and 

 3 ml of the preparation was drawn into each of 6 syringes which 

 already contained 7 ml of dilute (~50%) Riftia pachyptila blood 

 or vestimentiferan saline solution, variable concentrations of 

 sulfide (4-3000fM), and a marble for mixing the incubation 

 medium. To confirm that stimulated carbon fixation was through 

 autotrophic pathways, 10 mM D. L. glyceraldehyde (a feedback 

 inhibitor of RuBP carboxylase) was added to one syringe in each 

 series (Stokes and Walker 1972). 



The specific activity in each syringe was determined by 

 measuring the total inorganic C activity and the concentration 



179 



