Thiosulfate elimination and permeability in a sulfide-adapted marine invertebrate.

Oxidation of hydrogen sulfide to thiosulfate is one of the best-characterized mechanisms by which animals adapted to sulfide minimize its toxicity, but the mechanism of thiosulfate elimination in these animals has remained unclear. In this study, we examined the accumulation and elimination of thiosulfate in the sulfide-adapted marine worm Urechis caupo. The coelomic fluid of U. caupo exposed to 50-100 micromol L-1 sulfide in hypoxic seawater (Po2 ca. 10 kPa) accumulated (mean+/-SD) 132+/-41 micromol L-1 thiosulfate after 2 h, reaching 227+/-113 micromol L-1 after an additional 4 h in aerated, sulfide-free seawater. In whole-animal thiosulfate clearance studies, the rate of thiosulfate elimination from the coelomic fluid followed a single exponential time course with a half-life of 6 h. The thiosulfate permeability coefficient of isolated preparations mounted in diffusion chambers was 7.6x10-5+/-7. 7x10-5 cm s-1 for the hindgut and 5.5x10-7+/-2.7x10-7 cm s-1 for the body wall. These rates were independent of the direction of net efflux (mucosal-to-serosal or serosal-to-mucosal). Using a simple mathematical model of U. caupo that incorporates the thiosulfate permeability coefficients, the thiosulfate half-life was calculated to be 23 h without hindgut ventilation but less than 1 h with normal hindgut ventilation. Based on this information, we propose that passive thiosulfate diffusion across the hindgut is adequate to explain the observed rates of thiosulfate elimination.

Roots as a site of hydrogen sulfide uptake in the hydrocarbon seep vestimentiferan Lamellibrachia sp.

Vestimentiferan tubeworms have no mouth or gut, and the majority of their nutritional requirements are provided by endosymbiotic bacteria that utilize hydrogen sulfide oxidation to fix CO(2) into organic molecules. It has been assumed that all vestimentiferans obtain the sulfide, O(2) and CO(2) needed by the bacteria across the plume (gill) surface, but some live in locations where very little sulfide is available in the sea water surrounding the plume. We propose that at least some of these vestimentiferans can grow a posterior extension of their body and tube down into the sea-floor sediment, and that they can use this extension, which we call the 'root', to take up sulfide directly from the interstitial water. In this study of the vestimentiferan Lamellibrachia sp., found at hydrocarbon seeps in the Gulf of Mexico at depths of approximately 700 m, we measured seawater and interstitial sulfide concentrations in the hydrocarbon seep habitat, determined the structural characteristics of the root tube using transmission electron microscopy, characterized the biochemical composition of the tube wall, and measured the sulfide permeability of the root tube. We found that, while the sulfide concentration is less than 1 (micro)mol l(-)(1) in the sea water surrounding the gills, it can be over 1.5 mmol l(-)(1) at a depth of 10-25 cm in sediment beneath tubeworm bushes. The root tube is composed primarily of giant (&bgr;)-chitin crystallites (12-30 % of total mass) embedded in a protein matrix (50 % of total mass). Root tubes have a mean diameter of 1.4 mm, a mean wall thickness of 70 (micro)m and can be over 20 cm long. The tubeworm itself typically extends its body to the distal tip of the root tube. The root tube wall was quite permeable to sulfide, having a permeability coefficient at 20 degrees C of 0. 41x10(-)(3 )cm s(-)(1), with root tube being 2.5 times more permeable to sulfide than trunk tube of the same diameter. The characteristics of the root suggest that it reaches down to the higher sulfide levels present in the deeper sediment and that it functions to increase the surface area available for sulfide uptake in a manner analogous to a respiratory organ.

Neuromuscular sensitivity to hydrogen sulfide in the marine invertebrate Urechis caupo.

Hydrogen sulfide (HS) is a well-known inhibitor of aerobic respiration via its reversible binding of mitochondrial cytochrome c oxidase, but recent studies have suggested that HS may have other non-respiratory actions. We have studied the effects of HS on spontaneous and evoked contractions in vitro under hypoxic and anoxic conditions in nerve-muscle preparations from the echiuran worm Urechis caupo. Contraction amplitude in response to electric field stimulation under anoxic conditions was completely abolished by HS within minutes in a classic dose-response relationship (Kd=31 mmol l-1, r2=0.86). Exposure of body wall and esophagus to HS in vitro for up to 6 h demonstrated that the contraction amplitude and frequency of spontaneous activity were relatively insensitive to anoxia, but that the sensitivity to HS was similar to that seen in field-stimulated muscle (Kd=2.7-32 mmol l-1). The toxic effects of HS were reversible, with almost complete recovery under anoxic conditions within the first hour. These data indicate that HS at millimolar concentrations can directly inhibit muscle contraction. Although the mechanism of this action is unknown, it does not appear to involve metabolic pathways or oxygen transport.

Water lung and body wall contributions to respiration in an echiuran worm.

The modified hindgut of the echiuran marine worm Urechis caupo functions as a water lung and has been assumed to be a much more important respiratory surface than its thick, muscular body wall. We tested this assumption by measuring whole animal O2 consumption, hindgut O2 uptake, and hindgut ventilation activity simultaneously in unrestrained worms in artificial burrows from 25 to 300 Torr O2. Under experimental conditions the contribution of the hindgut to total O2 uptake is variable and strongly correlated to hindgut ventilatory activity. Over a PO2 range simulating that encountered in the natural environment, the hindgut accounts for approximately half of total O2 uptake on average. Under progressive hypoxic exposure total O2 consumption decreased by 50%, yet O2 conductance and extraction increased. The results suggest that the water lung function of the modified hindgut supplements O2 uptake across the body wall, and may be especially important during periods of high activity such as may occur during feeding and burrowing.

The Integument of the Marine Echiuran Worm Urechis caupo.

During low tide, the burrow water of the marine echiuran worm Urechis caupo becomes hypoxic, and hydrogen sulfide concentrations reach levels that would be toxic to most animals. Integument morphology in U. caupo is evaluated as an exchange surface and as a permeation barrier. Adaptive features include the rugose nature of the epidermis, which increases the surface area for oxygen uptake, and the thick muscular body wall, which provides a chief motive power in creating peristaltic movements along the body wall to ventilate the burrow. The epidermis is covered by a cuticle and contains two types of mucus-secreting cells: orthochromatic and metachromatic. Underlying connective tissue and three muscle layers form the bulk of the body wall. The integument does not present a significant structural barrier to permeation, although the mucus secreted by the epidermal cells may retard sulfide entry. Ultrastructural studies suggest three possible mechanisms that U. caupo may use to counteract the toxic effects of sulfide at the integumentary surface: metabolism of symbiotic bacteria embedded in the innermost cuticle layer and grouped together in the superficial epidermis, dying off of peripheral, sulfide-exposed cells, and oxidation of sulfide at specialized, ironrich, lysosomal organelles termed sulfide oxidizing bodies.

Oxygenation properties of the two co-occurring hemoglobins of the tube worm Riftia pachyptila.

Riftia pachyptila vascular blood and coelomic fluid contain two hemoglobin molecules that differ in their distribution and physical properties. The present study of the two isolated hemoglobins shows that both have an extremely high affinity for oxygen, but differ in their oxygenation characteristics. FI, the larger molecular weight (Mr) fraction (1,700,000), has a lower oxygen affinity, a well defined pH Bohr effect, and high cooperativity of oxygen binding. FII, the lower Mr fraction (400,000) has a higher oxygen affinity, no pH Bohr effect, and reduced cooperativity of oxygen binding. Both hemoglobins show marked effects of temperature on oxygen binding, and no effect of heme concentration or the presence of sulfide on oxygen affinity. The differences in the oxygenation properties and distribution of the two hemoglobins in the body fluids of Riftia pachyptila may allow them to play different roles in oxygen transport and storage for the animal which lives in the variable environment of the hydrothermal vents.

Oxygen binding characteristics of the hemocyanins of two deep-sea hydrothermal vent crustaceans.

Effects of pH and temperature on hemocyanin oxygen binding were examined using dialyzed blood from the hydrothermal vent crustaceans Bythograea thermydron (Brachyura) and Alvinocaris lusca (Caridea); blood samples were dialyzed against physiological salines having the same inorganic ion concentration as the blood. Hemocyanin of dialyzed B. thermydron blood demonstrated the highest O2 affinity at 10 degrees C (P50 = 1.8, 3.4 and 4.5 Torr at pH = 8.01, 7.7 and 7.4, respectively), with lower affinities at both higher and lower temperatures for all experimental pH values. A moderate Bohr effect (delta logP50/delta pH = -0.34 to -0.67) was found at all experimental temperatures (2-20 degrees C, pH 7.4-8.01), and a CO2 effect, independent of the pH Bohr effect, was measured at 5 degrees C. Hill coefficients varied widely (n50 = 0.95-3.95) and did not appear to vary with temperature or pH. The hemocyanin oxygen binding properties of B. thermydron blood are not affected by dialysis against salines having the same inorganic ion composition as the blood. Hemocyanin of dialyzed blood from A. lusca demonstrated a reverse temperature effect and a large Bohr effect (delta logP50/delta pH = -0.77 at 5 degrees C, pH 7.4-7.7). Functional aspects of oxygen binding are discussed in relation to the hydrothermal vent environment, and in the context of recent work on crustacean hemocyanins.

Sulfide Binding by the Blood of the Hydrothermal Vent Tube Worm Riftia pachyptila.

The blood of the deep-sea hydrothermal vent tube worm Riftia pachyptila Jones contains a sulfide-binding protein that appears to concentrate sulfide from the environment and may function for sulfide transport to the internal endosymbiotic bacteria contained within the coelomic organ, the trophosome.

Functional characteristics of the blood of the deep-sea hydrothermal vent brachyuran crab.

Hemocyanin in the whole blood of the hydrothermal vent brachyuran crab, Bythograea thermydron, has a moderate oxygen affinity (P(50) = 6.6 millimeters of mercury at 2.6 degrees C; pH 7.5), which unlike that of other hemocyanins is independent of temperature over the range 2 degrees to 30 degrees C; carbon dioxide and pH have independent effects on the oxygen affinity of this pigment. The pH effect on affinity is moderate (Deltalog P(50)/DeltapH = -0.34), whereas increased carbon dioxide, which can act both directly and by changing pH, has a much larger effect (Deltalog P(50)/DeltapH = -0.81). This blood has a moderately high degree of cooperativity (Hill cooperativity coefficient, n, was 2.8) and a large oxygen-carrying capacity for a crustacean (4.5 milliliters of oxygen per 100 milliliters of blood). These properties characterize an oxygen transport system whose function appears to be largely independent of the wide range of environmental conditions encountered around the vents.

Blood function in the hydrothermal vent vestimentiferan tube worm.

Extracellular hemoglobin in the whole blood of Riftia pachyptila has a high oxygen affinity (P50 = 1.8 millimeters of mercury at 3 degrees C), a moderate decrease in oxygen affinity at higher temperatures (P50 = 2.7 millimeters of mercury at 14 degrees C), a small effect of carbon dioxide on oxygen affinity (Delta log P50/Delta pH =-0.12), and a high oxygen carrying capacity (up to 11 milliliters of oxygen per 100 milliliters of blood). These characteristics are compatible with the high oxygen demand of chemoautotrophic metabolism in the variable vent environment.