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

Abstract

The vestimentiferan tubeworm Riftia pachyptila derives most or all of its nutrition from intracellular chemosynthetic bacterial symbionts. Because purified preparations of symbionts respire nitrate, possibly nitrite, and oxygen, host transport of nitrate is a topic of interest. In the present study, we have developed a nitrate detection assay that utilizes a nitrite reductase-deficient Escherichia coli strain for the reduction of nitrate to nitrite, which is then determined spectrophotometrically. Nitrate and nitrite concentrations were measured in the blood and coelomic fluids of R. pachyptila collected from hydrothermal vent sites at 9°N and 13°N. The blood was shown to have nitrate concentrations up to one hundred times that of ambient sea water (40 μM). Blood nitrate levels reached concentrations of > 1 mM, while nitrite was measured in the range of 400-700 μM. The concentrations of nitrate and nitrite in the coelomic fluids were 150-240 μM and <20 μM, respectively. The nitrate determination technique we present here is simple, applicable for laboratory and shipboard use on sea water or biological fluids, and works reliably within the 0.5 to 2000 μM range.