Ecological Physiology and Biochemistry of Sulfide Acquisition by Two Hydrocarbon Seep Vestimentiferans, Lamellibrachia luymesi and Seepiophila jonesi

open_access
Open Access
Author:
Freytag, John Karl
Graduate Program:
Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
July 23, 2003
Committee Members:
Charles Raymond Fisher Jr., Committee Chair/Co-Chair
James Harold Marden, Committee Member
Roger Tai Koide, Committee Member
Michael Allan Arthur, Committee Member
James J Childress, Committee Member
Keywords:
sulfide
vestimentiferan
hemoglobin
Riftia
Lamellibrachia
Seepiophila
Abstract:
Two species of vestimentiferan tubeworm, Lamellibrachia luymesi and Seepiophila jonesi, co-occur in aggregations at northern Gulf of Mexico cold hydrocarbon seep sites. Like all vestimentiferans, L. luymesi and S. jonesi obtain nutrition from sulfide-oxidizing chemoautotrophic bacterial endosymbionts that must be supplied with sulfide, oxygen, and carbon dioxide. Results from previous studies that examined the environmental sulfide chemistry of northern Gulf of Mexico hydrocarbon seeps suggested that the ecological physiology of seep tubeworms was not analogous to that of the hydrothermal vent tubeworm, Riftia pachyptila, which obtains sulfide, oxygen, and carbon dioxide across the anterior plume portion of its body. The focus of this study was to better understand the physiological ecology of environmental sulfide acquisition of L. luymesi and S. jonesi. Whole animal respiration studies were conducted using split-vessel respiration chambers built specifically for this series of experiments. Methods for sulfide equilibrium dialysis experiments were determined and utilized to estimate the sulfide-binding characteristics of the intact fluids and component hemoglobins of L. luymesi and S. jonesi. L. luymesi and S. jonesi grow a posterior extension of their tube and tissue, termed a “root,” down into sulfidic sediments below the point of original attachment. Preliminary blood sulfide uptake experiments confirmed that sulfide uptake across the posterior portions of live L. luymesi can occur. Sulfide was not detectable in the blood of any control animals that did not have their roots exposed to sulfide (detection limit of 3.0 µM) but was present in the blood of experimental animals that had their roots exposed to 500 µM sulfide (152 and 170 µM). Split-vessel respiration experiments which exposed the root portions of L. luymesi to sulfide concentrations between 51 and 561 µM demonstrated that L. luymesi can utilize their roots as a respiratory surface to acquire sulfide at an average rate of 4.1 µmoles*g-1*h-1. Net dissolved inorganic carbon uptake across the plume of the tubeworms was shown to occur in response to exposure of the posterior, root portion of the worms to sulfide, demonstrating that sulfide acquisition by roots of the seep vestimentiferan L. luymesi can be sufficient to fuel net autotrophic total dissolved inorganic carbon uptake. Vestimentiferan vascular blood and coelomic fluid contain giant extracellular hemoglobins (Hbs; a 3,500 kDa and a 400 kDa Hb in the vascular blood, and a different 400 kDa Hb in coelomic fluid) that reversibly and simultaneously bind large quantities of hydrogen sulfide and oxygen at binding sites unique for each chemical species. These Hbs are fundamental to the uptake, accumulation, transportation, and delivery of both sulfide and oxygen to their endosymbionts. Sulfide-binding dialysis experiments were conducted in order to estimate the sulfide-binding affinity and capacity for the purified R. pachyptila 3,500 kDa Hb and a mixture of 400 kDa Hbs from vascular and coelomic fluids. Under experimental conditions, the 3,500 kDa Hb bound 2.2 moles sulfide per mole of heme when saturated. Half-saturation occurred at 5.2µM SH2S (the sum of H2S, HS-, and S2- ) for this Hb. The 400 kDa Hbs were able to bind 0.47 moles sulfide per mole of heme when saturated, and 50% saturation occurred at 5.4µM SH2S. The very similar sulfide affinities of the 3,500 kDa and 400 kDa Hb fractions would allow bi-directional exchange and storage of sulfide in both fluid compartments and is consistent with the proposed role of the coelomic fluid as a temporary storage reservoir for sulfide. During this series of experiments, significant limitations in the methods commonly used in sulfide-binding studies were found. At equilibrium, sulfide concentrations inside small volume dialysis bags were not equal to external dialysate concentrations, and the effect varied with the bag volume. Sulfide-binding by purified 3,500 kDa and 400 kDa Riftia pachyptila Hb fractions was also positively correlated to heme concentration. Sulfide binding per heme did not increase significantly above concentrations of 0.4 mM heme in the experiments with the 400 kDa Hbs. However, sulfide bound per heme by the 3,500 kDa Hb increased significantly over the full range of heme concentrations tested (0.15 to 1.8 mM). The mechanism for increased sulfide-binding at increased heme concentrations has not been determined. Utilizing an improved methodology resulting from the sulfide-binding experiments with R. pachyptila fluid and hemoglobins, experiments were conducted to estimate the sulfide-binding affinity and capacity for purified 3,500 kDa Hbs and mixtures of 400 kDa Hbs from the vascular and coelomic fluids of L. luymesi and S. jonesi. In addition, the heme concentrations and relative Hb abundance of intact vascular blood and coelomic fluids and the sulfide-binding characteristics of the component Hbs were determined for both L. luymesi and S. jonesi. Results from sulfide-binding experiments show that the 3,500 kDa Hb from the fluids of S. jonesi has a high affinity for sulfide (C50 value of 8.8µM) while the 3,500 kDa Hb from the fluids of L. luymesi has only a moderate affinity for sulfide (C50 value of 96µM). S. jonesi has elevated fluid heme concentrations that may facilitate the survival of individuals in environments where they are exposed to low concentrations of oxygen and/or short periods without any oxygen. The high affinity of the predominant hemoglobin in the vascular fluid of S. jonesi for sulfide suggests that large S. jonesi may be capable of acquiring sulfide with the anterior plume portion of its body from low concentration pools found just above the sediment-water interface. The low affinity of the most prevalent hemoglobin in the vascular fluid of L. luymesi for sulfide suggests that large L. luymesi are not likely able to use their plumes for sulfide uptake and likely depend upon the root portion of the body and tube for sulfide acquisition. The sulfide-binding data for the L. luymesi 3,500 kDa Hb suggests that L. luymesi may have a 3,500 kDa Hb with multiple sulfide-binding mechanisms that have different sulfide capacity and C50 values (one lower and one higher) or express two different 3,500 kDa Hbs with different sulfide-binding characteristics. Together these data have fundamentally changed our model for the ecological physiology of seep vestimentiferans. Although closely related to hydrothermal vent tubeworms, L. luymesi and S. jonesi do not acquire metabolites from the environment in the same way as R. pachyptila. We are now beginning to understand how sulfide, carbon dioxide, and oxygen can be acquired by L. luymesi and S. jonesi.