with methanol dehydrogenase, the second enzyme in the pathway 

 for the oxidation of methane. It is also the only species which 

 shows methane consumption, and as Fisher et al. (1987) have 

 shown the rate of consumption is proportional to the activity of 

 this enzyme in individual mussels. The seep mussel is also the 

 only species among those examined whose bacteria have the 

 internal membranes characteristic of Type I methanotrophic 

 bacteria (Anthony 1982). In addition the seep mussel gills lack 

 the enzymes chacteristic of sulfur oxidation (adenosine 

 triphosphate sulfurylase and adenosine-5 ' -phosphosulf ate 

 reductase), lack elemental sulfur, and have only trace activities 

 of RuBP carboxylase (an enzyme characteristic of autotrophic 

 carbon fixation); these factors indicate that the seep mussel 

 symbionts are not sulfur-oxidizing chemoautotrophs . 



The other three bivalves and the two vestimentiferans from 

 the seeps appear to harbor sulfur-oxidizing chemoautolithotrophic 

 symbionts. The enzyme activities, the presence of elemental 

 sulfur, carbon fixation stimulated by sulfide, and electron 

 microscopy provide evidence that both vestimentiferans and the 

 lucinid clam, Pseudomiltha sp., contain chemoautotrophic, sulfur 

 bacterial symbionts. The evidence for the vesicomyid clams is 

 not as conclusive since we have not yet analyzed the enzyme 

 activities in tissues frozen alive in liquid nitrogen. The 

 absence of specific enzyme activities is thus of little 

 significance. Nonetheless, the high level of elemental sulfur 

 in the gills of C. ponderosa and the high levels of ATP 

 sulfurylase in V. cordata gills suggest that sulfur oxidizing 

 symbionts are present in the living clams. The sulfide oxidase 

 activities in all animals assayed are at the level expected for 

 invertebrates exposed to a sulfide environment (Powell and 

 Somero 1986). 



The absence of methane consumption in live animals and the 

 absence of methanol dehydrogenase activity in the symbiont 

 containing tissues clearly indicates that the three vent species 

 (shown in Table 1) do not harbor methanotrophic symbionts. The 

 presence of substantial activities of RuBP carboxylase and ATP 

 sulfurylase, elemental sulfur in symbiont containing tissues, and 

 sulfide stimulation of carbon fixation in these same tissues 

 leaves little doubt that these symbionts are 

 chemoautolithotrophic sulfur oxidizers. The mussel B. 

 thermophilus is however more problematic since it lacks a strong 

 indication of sulfur chemoautotrophy and is clearly not hosting 

 methanotrophs (Fisher et al . 1987). 



DISCUSSION 



The data presented here demonstrate the necessity of using as 

 many criteria as possible to characterize the nutrition of 

 symbionts in chemoautotrophic symbioses in the absence of the 

 ability to culture the symbionts. Any one or even several 



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