The nutrition of these symbioses can be approached from 

 several perspectives. First there is the question of the energy 

 and carbon sources of the symbionts. Second is the interaction 

 of the hosts with the inorganic substrates including the 

 possible derivation of energy from inorganic substrates by the 

 hosts (Powell and Somero 1986). Third is the degree to which 

 the hosts obtain reduced carbon compounds from their symbionts 

 and from their environment. The nature of the symbiont nutrition 

 can be inferred from a variety of information including assays 

 for key enzymes in the pathways of oxidation of reduced inorganic 

 compounds and the fixation of inorganic carbon, ratios of stable 

 carbon isotopes, autotrophic C02 fixation, elemental sulfur 

 contents and symbiont ultrastructure. Additional information 

 about the symbiont nutrition as well as its relationship to the 

 host nutrition can be obtained from studies of the metabolism and 

 growth of the host with symbiont energy and carbon sources. 

 This study describes our efforts to date to determine the 

 nutritional basis of the major sessile invertebrates living 

 around the hydrocarbon seeps. 



METHODS 



The animals studied were collected with the manipulator of 

 the Johnson Sea Link submersible. They were brought to the 

 surface in a closed box which protected them from the warm 

 surface temperatures. Once at the surface the animals were kept 

 alive in cold seawater until used. Samples to be frozen were 

 placed in cryovials and then in liquid nitrogen. 



Enzyme Assays 



Enzyme assays were done on samples which had been frozen in 

 liquid nitrogen and stored at -70° C. The weighed tissue was 

 homogenized with a ground-glass tissue-grinder in 0.2mM Tris/HCl 

 (pH 7.5) with 1% Triton X100 (1:7 tissue : buf fer) . This crude 

 homogenate was assayed for RuBP carboxylase activity by the 

 14 C-incorporation method (Wishnick and Lane, 1971) as modified 

 by Felbeck (1981) and methanol dehydrogenase activity by the 

 spectrophotometric method of Anthony and Zatman (1965). ATP 

 sulfurylase and APS reductase activities were assayed using the 

 methods described by Felbeck (1981). 



Histology 



For transmission electron microscopy pieces of gill from 

 freshly recovered animals were fixed in 3% glutaraldehyde in 0.1M 

 phosphate-buffered 0.35M sucrose pH 7.3, and stored in this 

 fixative at 4x C for up to two weeks. The tissues were then 

 washed in buffered sucrose, post-fixed in 1% osmium (on ice) for 

 1 hour, dehydrated through a graded ethanol series, and embedded 

 in Spurrs embedding medium. Thin sections were cut using an LKB 



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