Determination of nitrate in the blood of the hydrothermal vent tubeworm Riftia pachyptila using a bacterial nitrate reduction assay

The vestimentiferan tubeworm Riftia pachyptila derives most or all of its n utrition from intracellular chemosynthetic bacterial symbionts. Because pur ified preparations of symbionts respire nitrate, possibly nitrite, and oxyg en, host transport of nitrate is a topic of interest. In the present study, we have developed a nitrate detection assay that utilizes a nitrite reduct ase-deficient Escherichia coli strain for the reduction of nitrate to nitri te, which is then determined spectrophotometrically. Nitrate and nitrite co ncentrations were measured in the blood and coelomic fluids of R. pachyptil a collected from hydrothermal vent sites at 9 degrees N and 13 degrees N. T he blood was shown to have nitrate concentrations up to one hundred times t hat of ambient sea water (40 mu M). Blood nitrate levels reached concentrat ions of >1 mM, while nitrite was measured in the range of 400-700 mu M. The concentrations of nitrate and nitrite in the coelomic fluids were 150-240 mu M and <20 mu M, respectively. The nitrate determination technique we pre sent here is simple, applicable for laboratory and shipboard use on sea wat er or biological fluids, and works reliably within the 0.5 to 2000 mu M ran ge. (C) 1998 Elsevier Science Ltd. All rights reserved.