The metabolic demands of endosymbiotic chemoautotrophic metabolism on host physiological capacities.

While chemoautotrophic endosymbioses of hydrothermal vents and other reducing environments have been well studied, little attention has been paid to the magnitude of the metabolic demands placed upon the host by symbiont metabolism and the adaptations necessary to meet such demands. Here we make the first attempt at such an evaluation, and show that moderate to high rates of chemoautotrophic or methanotrophic metabolism impose oxygen uptake and proton equivalent elimination demands upon the hosts that are much higher than is typical for the non-symbiotic annelid, bivalve and gastropod lineages to which they are related. The properties of the hosts are described and compared to determine which properties are associated with and predictive of the highest rates. We suggest that the high oxygen demand of these symbionts is perhaps the most limiting flux for the symbioses. Among the consequences of such demands has been the widespread presence of circulating and/or tissue hemoglobins in these symbioses that are necessary to support high metabolic rates in thioautotrophic endosymbioses. We also compare photoautotrophic with chemoautotrophic and methanotrophic endosymbioses to evaluate the differences and similarities in physiologies. These analyses suggest that the high demand for oxygen by chemoautotrophic and methanotrophic symbionts is likely a major factor precluding their endosymbiosis with cnidarians.

Effects of metabolite uptake on proton-equivalent elimination by two species of deep-sea vestimentiferan tubeworm, Riftia pachyptila and Lamellibrachia cf luymesi: proton elimination is a necessary adaptation to sulfide-oxidizing chemoautotrophic symbionts.

Intracellular symbiosis requires that the host satisfy the symbiont's metabolic requirements, including the elimination of waste products. The hydrothermal vent tubeworm Riftia pachyptila and the hydrocarbon seep worm Lamellibrachia cf luymesi are symbiotic with chemolithoautotrophic bacteria that produce sulfate and protons as end-products. In this report, we examine the relationship between symbiont metabolism and host proton equivalent elimination in R. pachyptila and L. cf luymesi, and the effects of sulfide exposure on proton-equivalent elimination by Urechis caupo, an echiuran worm that lacks intracellular symbionts (for brevity, we will hereafter refer to proton-equivalent elimination as 'proton elimination'). Proton elimination by R. pachyptila and L. cf luymesi constitutes the worms' largest mass-specific metabolite flux, and R. pachyptila proton elimination is, to our knowledge, the most rapid reported for any metazoan. Proton elimination rates by R. pachyptila and L. cf luymesi correlated primarily with the rate of sulfide oxidation. Prolonged exposure to low environmental oxygen concentrations completely inhibited the majority of proton elimination by R. pachyptila, demonstrating that proton elimination does not result primarily from anaerobic metabolism. Large and rapid increases in environmental inorganic carbon concentrations led to short-lived proton elimination by R. pachyptila, as a result of the equilibration between internal and external inorganic carbon pools. U. caupo consistently exhibited proton elimination rates 5-20 times lower than those of L. cf luymesi and R. pachyptila upon exposure to sulfide. Treatment with specific ATPase inhibitors completely inhibited a fraction of proton elimination and sulfide and inorganic carbon uptake by R. pachyptila, suggesting that proton elimination occurs in large part via K(+)/H(+)-ATPases and Na(+)/H(+)-ATPases. In the light of these results, we suggest that protons are the primary waste product of the symbioses of R. pachyptila and L. cf luymesi, and that proton elimination is driven by symbiont metabolism, and may be the largest energetic cost incurred by the worms.

A paradox resolved: sulfide acquisition by roots of seep tubeworms sustains net chemoautotrophy.

Vestimentiferan tubeworms, symbiotic with sulfur-oxidizing chemoautotrophic bacteria, dominate many cold-seep sites in the Gulf of Mexico. The most abundant vestimentiferan species at these sites, Lamellibrachia cf. luymesi, grows quite slowly to lengths exceeding 2 meters and lives in excess of 170-250 years. L. cf. luymesi can grow a posterior extension of its tube and tissue, termed a "root," down into sulfidic sediments below its point of original attachment. This extension can be longer than the anterior portion of the animal. Here we show, using methods optimized for detection of hydrogen sulfide down to 0.1 microM in seawater, that hydrogen sulfide was never detected around the plumes of large cold-seep vestimentiferans and rarely detectable only around the bases of mature aggregations. Respiration experiments, which exposed the root portions of L. cf. luymesi to sulfide concentrations between 51-561 microM, demonstrate that L. cf. luymesi use their roots as a respiratory surface to acquire sulfide at an average rate of 4.1 micromol x g(-1) x 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. cf. luymesi can be sufficient to fuel net autotrophic total dissolved inorganic carbon uptake.

Reduced enzymatic antioxidative defense in deep-sea fish.

Oxygen, while being an obligate fuel for aerobic life, has been shown to be toxic through its deleterious reactive species, which can cause oxidative stress and lead ultimately to cell and organism death. In marine organisms, reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide, are generated within respiring cells and tissues and also by photochemical processes in sea water. Considering both the reduced metabolic rate of nektonic organisms thriving in the deep sea and the physico-chemical conditions of this dark, poorly oxygenated environment, the meso- and bathypelagic waters of the oceans might be considered as refuges against oxidative dangers. This hypothesis prompted us to investigate the activities of the three essential enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX) constitutive of the antioxidative arsenal of cells in the tissues of 16 species of meso- and bathypelagic fishes occurring between the surface and a depth of 1300 m. While enzymatic activities were detected in all tissues from all species, the levels of SOD and GPX decreased in parallel with the exponential reduction in the metabolic activity as estimated by citrate synthase activity. In contrast, CAT was affected neither by the metabolic activity nor by the depth of occurrence of the fishes. High levels of metabolic and antioxidative enzymes were detected in the light organs of bioluminescent species. The adjustment of the activity of SOD and GPX to the decreased metabolic activity associated with deep-sea living suggests that these antioxidative defense mechanisms are used primarily against metabolically produced ROS, whereas the maintenance of CAT activity throughout all depths could be indicative of another role. The possible reasons for the occurrence of such a reduced antioxidative arsenal in deep-sea species are discussed.

Polypeptide chain composition diversity of hexagonal-bilayer haemoglobins within a single family of annelids, the alvinellidae.

Following previous analysis of the structure of Alvinella pompejana heaxagonal-bilayer haemoglobin (HBL Hb) [1], we report in this paper the structure of three other HBL Hbs belonging to Alvinella caudata, Paralvinella grasslei and Paralvinella palmiformis, members of the Alvinellidae, annelid family strictly endemic to deep-sea hydrothermal vents located on the ridge crests in the Pacific ocean. The multi-angle laser light scattering (MALLS) and fast protein liquid chromatography (FPLC) analysis revealed a broad range of molecular masses for the extracellular Hb molecules, 3517 +/- 14 kDa (A. caudata), 3822 +/- 28 kDa (P. grasslei) and 3750 +/- 150 kDa (P. palmiformis). Native and derivative Hbs (reduced, carbamidomethylated and deglycosylated) were analysed by electrospray ionization mass spectroscopy (ESI-MS) and the data was processed by the maximum entropy deconvolution system (MaxEnt). The most important difference between alvinellid HBL Hbs was the variation in their composition, from two to four monomeric globin chains, and from one to four linker chains. Therefore, despite the fact that all these species belong to a single family, notable differences in the polypeptide chain composition of their HBL Hbs were observed, probably accounting for their different functional properties as previously reported by this group Toulmond, A., El Idrissi Slitine, F., De Frescheville, J. & Jouin, C. (1990) Biol. Bull. 179, 366-373.

Fate of nitrate acquired by the tubeworm Riftia pachyptila.

The hydrothermal vent tubeworm Riftia pachyptila lacks a mouth and gut and lives in association with intracellular, sulfide-oxidizing chemoautotrophic bacteria. Growth of this tubeworm requires an exogenous source of nitrogen for biosynthesis, and, as determined in previous studies, environmental ammonia and free amino acids appear to be unlikely sources of nitrogen. Nitrate, however, is present in situ (K. Johnson, J. Childress, R. Hessler, C. Sakamoto-Arnold, and C. Beehler, Deep-Sea Res. 35:1723-1744, 1988), is taken up by the host, and can be chemically reduced by the symbionts (U. Hentschel and H. Felbeck, Nature 366:338-340, 1993). Here we report that at an in situ concentration of 40 microM, nitrate is acquired by R. pachyptila at a rate of 3.54 micromol g(-1) h(-1), while elimination of nitrite and elimination of ammonia occur at much lower rates (0. 017 and 0.21 micromol g(-1) h(-1), respectively). We also observed reduction of nitrite (and accordingly nitrate) to ammonia in the trophosome tissue. When R. pachyptila tubeworms are exposed to constant in situ conditions for 60 h, there is a difference between the amount of nitrogen acquired via nitrate uptake and the amount of nitrogen lost via nitrite and ammonia elimination, which indicates that there is a nitrogen "sink." Our results demonstrate that storage of nitrate does not account for the observed stoichiometric differences in the amounts of nitrogen. Nitrate uptake was not correlated with sulfide or inorganic carbon flux, suggesting that nitrate is probably not an important oxidant in metabolism of the symbionts. Accordingly, we describe a nitrogen flux model for this association, in which the product of symbiont nitrate reduction, ammonia, is the primary source of nitrogen for the host and the symbionts and fulfills the association's nitrogen needs via incorporation of ammonia into amino acids.

Light-limitation on predator-prey interactions: consequences for metabolism and locomotion of deep-sea cephalopods.

The present study attempts to correlate the metabolism and locomotory behavior of 25 species of midwater Cephalopoda from California and Hawaii with the maximal activities of key metabolic enzymes in various locomotory muscle tissues. Citrate synthase (CS) and octopine dehydrogenase (ODH) activities were used as indicators of aerobic and anaerobic metabolic potential respectively. CS activity in mantle muscle is highly correlated with whole-animal rates of oxygen consumption, whereas ODH activity in mantle muscle is significantly correlated with a species' ability to buffer the acidic end-products of anaerobic metabolism. Both CS and ODH activities in mantle muscle declined strongly with a species' habitat depth. For example, CS and ODH activities ranged respectively from 0.04 units g(-1) and 0.03 units g(-1) in the deep-living squid Joubiniteuthis portieri, to 8.13 units g(-1) and 420 units g(-1) in the epipelagic squid Sthenoteuthis oualaniensis. The relationships between enzymatic activities and depth are consistent with similar patterns observed for whole-animal oxygen consumption. This pattern is believed to result from a relaxation, among deep-living species, in the need for strong locomotory abilities for visual predator/prey interactions; the relaxation is due to light-limitation in the deep sea. Intraspecific scaling patterns for ODH activities may, for species that migrate ontogenetically to great depths, reflect the counteracting effects of body size and light on predator-prey detection distances. When scaled allometrically, enzymatic activities for the giant squid, Architeuthis sp., suggest a fairly active aerobic metabolism but little burst swimming capacity. Interspecific differences in the relative distributions of enzymatic activities in fin, mantle, and arm tissue suggest an increased reliance on fin and arm muscle for locomotion among deep-living species. We suggest that, where high-speed locomotion is not required, more efficient means of locomotion, such as fin swimming or medusoid arm propulsion, are more prevalent.

The ionic composition of the hydrothermal vent tube worm Riftia pachyptila: evidence for the elimination of SO2-4SO and H+ and for a Cl-/HCO-3HCO shift.

Riftia pachyptila is one of the most specialized invertebrate hosts of chemoautotrophic symbionts. Crucial to the functioning of this symbiosis is how these worms cope with fluctuating ion concentrations. Internal sulfate levels in R. pachyptila appear comparable with other benthic marine invertebrates, despite the production of sulfate internally by means of the bacterial oxidation of hydrogen sulfide, suggesting that these worms are able to eliminate sulfate effectively. Internal chloride levels appear comparable; however, coelomic fluid chloride levels decrease significantly as the amount of coelomic fluid bicarbonate increases, demonstrating a 1:1 stoichiometry. We believe this shift in chloride, out of the body fluids, is needed to compensate for changes in electrochemical balance caused by the large increase (up to and greater than 60 mmol L-1) in negatively charged bicarbonate. Riftia pachyptila fits the general pattern of monovalent ion concentrations that is seen in other benthic marine invertebrates, with a high [Na+] : [K+] ratio extracellularly and low [Na+] : [K+] ratio intracellularly. Extracellular pH values of 7.38+/-0.03 and 7.37+/-0. 04 for coelomic fluid and vascular blood, respectively, as well as intracellular pH values of 7.37+/-0.04 and 7.04+/-0.05 for plume and trophosome tissue, respectively, were measured. On the basis of significant decreases in extracellular pH and, in some cases, Na+ and K+, in worms exposed to carbonyl cyanide m-chlorophenylhydrazone, sodium vanadate, and N-ethylmaleimide, we suggest that high concentrations of H+-ATPases, perhaps Na+/H+- or K+/H+-ATPases, are involved in H+ elimination in these animals.

Physiological Functioning of Carbonic Anhydrase in the Hydrothermal Vent Tubeworm Riftia Pachyptila.

On the basis of our experiments, it is clear that carbonic anhydrase (CA) plays an important role in the CO2-concentrating mechanisms in Riftia pachyptila. Plume tissue from freshly collected animals had the highest CA activity, 253.7 +/- 36.0 {mu}mol CO2 min-1 g-1 wet wt, and trophosome activity averaged 109.4 +/- 17.9 {mu}mol CO2 min-1 g-1 wet wt. Exposure of living worms to ethoxyzolamide, a carbonic anhydrase inhibitor, resulted in a 99% decrease in CA activity (from 103.9 +/- 38.6 to 0.7 +/- 0.2 {mu}mol CO2 min-1 g-1 wet wt in the plume tissue and 57.6 +/- 17.9 to 0.04 +/- 0.11 {mu}mol CO2 min-1 g-1 wet wt in the trophosome) and essentially a complete cessation of {Sigma}CO2 uptake. High concentrations of CA appear to facilitate the equilibration between inorganic carbon (Ci) in the external and internal environments, greatly enhancing the diffusion of CO2 into the animal. In summary, R. pachyptila demonstrates very effective acquisition of inorganic carbon from the environment, thereby providing the symbionts with large amounts of CO2. This effective acquisition is made possible by three factors: extremely effective pH regulation, a large external pool of CO2, and, described in this paper, high levels of carbonic anhydrase.

S-Sulfohemoglobin and disulfide exchange: the mechanisms of sulfide binding by Riftia pachyptila hemoglobins.

The deep sea hydrothermal tube worm Riftia pachyptila possesses a multihemoglobin system with three different extracellular hemoglobins (Hbs; V1, V2, and C1): two dissolved in the vascular blood, V1 and V2, and one in the coelomic fluid, C1. V1 consists of four heme-containing chains and four linker chains. The globin chains making up V2 and C1 are, with one exception, common to V1. Remarkably these Hbs are able to bind oxygen and sulfide simultaneously and reversibly at two different sites. Two of the globin chains found in these three Riftia Hbs possess one free Cys residue and for at least one of the globins, the b-Cys75 is conserved among vestimentifera (Lamellibrachia sp.) and pogonophora (Oligobrachia mashikoi). By selectively blocking the free Cys with N-ethylmaleimide and using electrospray ionization mass spectrometry experiments, we show that these Cys residues are involved in sulfide binding by Riftia Hbs. Moreover, we also demonstrate that the larger V1 Hb can form persulfide groups on its linker chains, a mechanism that can account for the higher sulfide-binding potential of this Hb.

Life at stable low oxygen levels: adaptations of animals to oceanic oxygen minimum layers.

Zones of minimum oxygen level are found at intermediate depths in most of the world's oceans and, although the oxygen partial pressure in some of these 'oxygen minimum layers' is only a fraction of a kilopascal, populations of pelagic metazoans exist there. These oxygen minimum layers are areas of the water column and the associated benthos with stable conditions of continuously low oxygen level and low temperature at intermediate depths (400-1000 m depth) over vast areas. Off California, where PO2 at the oxygen minimum is 0.8 kPa, there are abundant populations of animals both in the water column and on the bottom. Farther to the south in the eastern tropical Pacific, oxygen partial pressures of less than approximately 0.4 kPa result in very low biomasses and diversity of animals at minimum layer depths. At the minimum oxygen levels found off California, most animals which inhabit the minimum zones appear to support their routine metabolic demands via aerobic metabolism. They do this by being very effective at removing oxygen from water. Among the adaptations of pelagic crustaceans to these conditions are: (1) enhanced ventilatory abilities, (2) enhanced percentage removal of O2 from the ventilatory stream, (3) large gill surface areas, (4) short diffusion distances from the water to the blood, and (5) hemocyanin respiratory proteins with a very high affinity for O2, high cooperativity and large Bohr effects. The lower O2 consumption rates of many deeper-living species are also functionally adaptive in that they facilitate aerobic survival at low PO2. However, they are not adaptations to the minimum layer, since similarly low rates are found in the same and comparable species living at the same depths in regions without well-developed minima, and these animals are unable to survive at the low PO2 values of the minima. While anaerobic metabolism may be important for metabolic rates above the routine level for most animals in the minimum layer, there is little evidence for the use of sustained anaerobiosis in the species studied. In summary, given the stable presence of very low O2 levels in the minima, the primary adaptations of animals living within them are those that support aerobic metabolism by giving the animals remarkable abilities to extract O2 from water. These abilities are notably better than those of animals adapted to unstable hypoxic environments, such as intertidal mudflats, while the latter animals rely to a much greater extent on anaerobiosis and perhaps on metabolic suppression to survive periods of anoxia.

Primary structure of the common polypeptide chain b from the multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila: an insight on the sulfide binding-site.

The deep-sea tube worm Riftia pachyptila Jones possesses a multi-hemoglobin system with three different extracellular Hbs: two dissolved in the vascular blood, V1 (ca. 3,500 kDa) and V2 (ca. 400 kDa), and one in the coelomic fluid, C1 (ca. 400 kDa). V1 Hb consists of four heme-containing, globin chains (b-e) and four linker chains (L1-L4). V2 and C1 Hbs are exclusively built from globin chains, six for V2 (a-f) and five for C1 (a-e). The complete amino acid sequence of the isolated monomeric globin chain b, common to all Riftia Hbs, has been determined by automated Edman degradation sequencing of the peptides derived by digestion with trypsin, chymotrypsin, thermolysin, and CNBr. This polypeptide chain is composed of 144 amino acid residues, providing a M(r) of 16, 135.0 Da. Moreover, the primary sequence of chain b revealed 3 Cys residues at position 4, 75, and 134. Cys-4 and Cys-134 are located at positions where an intra-chain disulfide bridge is formed in all annelid, vestimentiferan, or pogonophoran chains, but Cys-75 is located at a unique position only found in three globin chains belonging to Lamellibrachia and Oligobrachia, a vestimentiferan and a pogonophoran. In both groups, Hbs can bind sulfide reversibly to fuel the chemosynthetic process of the symbiotic bacteria they harbor. Sulfide-binding experiments performed on purified Hb fractions (i.e., V1, V2, and C1 Hbs) suggest that free Cys residues on globin chains, and the numerous Cys found in linker chains, as determined previously by ESI-MS, may be the sulfide binding-sites. Blocking the free Cys by N-ethylmaleimide, we confirmed that free cysteines were involved in sulfide-binding but did not account for the whole sulfide-binding capacity of V1 Hb. Furthermore, a phylogenetic tree was constructed from 18 globin-like chains of annelid, vestimentiferan, and pogonophoran extracellular Hbs to clarify the systematic position of tubeworms. Riftia chain b clearly belongs to the "strain A" family with 30 to 80% identity with the other sequences analyzed. Its position in the tree confirmed a close relationship between vestimentiferan, pogonophoran, and annelid Hbs.

Sulfide acquisition by the vent worm Riftia pachyptila appears to be via uptake of HS-, rather than H2S.

Deep-sea hydrothermal vents are home to a variety of invertebrate species, many of which host chemosynthetic bacteria in unusual symbiotic arrangements. The vent tubeworm Riftia pachyptila (Vestimentifera) relies upon internal chemolithoautotrophic bacterial symbionts to support its large size and high growth rates. Because of this, R. pachyptila must supply sulfide to the bacteria, which are far removed from the external medium. Internal H2S ([H2S+HS-+S2-]) can reach very high levels in R. pachyptila (2-12mmoll-1 in the vascular blood), most of which is bound to extracellular hemoglobins. The animal can potentially take up sulfide from the environment via H2S diffusion or via mediated uptake of HS-, or both. It was expected that H2S diffusion would be the primary sulfide acquisition mechanism, paralleling the previously demonstrated preferential uptake of CO2. Our data show, however, that the uptake of HS- is the primary mechanism used by R. pachyptila to obtain sulfide and that H2S diffusion into the worm apparently proceeds at a much slower rate than expected. This unusual mechanism may have evolved because HS- is less toxic than H2S and because HS- uptake decouples sulfide and inorganic carbon acquisition. The latter occurs via the diffusion of CO2 at very high rates due to the maintenance of an alkaline extracellular fluid pH. H2S accumulation is limited, however, to sulfide that can be bound by the hemoglobins, protecting the animal from sulfide toxicity and the symbionts from sulfide inhibition of carbon fixation.

Decline in Pelagic Cephalopod Metabolism With Habitat Depth Reflects Differences in Locomotory Efficiency.

The metabolic rates of 33 species of pelagic cephalopods from California and Hawaii were measured and correlated with minimum depth of occurrence. Mean metabolic rates ranged from 0.07 {mu}mol O2g-1 h-1 for the deep-living vampire squid, Vampyroteuthis infernalis, to 8.79 {mu}mol O2 g-1 h-1 for Gonatus onyx, a vertically migrating squid. An individual of V. infernalis, which lives within the oxygen minimum layer off California, had the lowest mass-specific metabolic rate ever measured for a cephalopod (0.02 {mu}mol O2g-1 h-1, 1050 g wet weight). For species collected in sufficient quantity and size range, metabolism was related to body size. Critical partial pressures of oxygen (Pc) were determined for Hawaiian and Californian cephalopods. Pc values for Hawaiian animals were considerably higher than for those taken off California, a trend that corresponds to the higher levels of environmental oxygen in the Hawaiian waters. Buffering capacity ({beta}) of mantle muscle, assayed in eight cephalopod species, was used to estimate the capacity for glycolytic energy production. Mean {beta} ranged from 1.43 slykes for a bathypelagic octopod, Japetella heathi, to 77.08 slykes for an epipelagic squid. Sthenoteuthis oualaniensis. Significant declines with increasing depth of occurrence were observed for both metabolism and {beta}. The decline in metabolic parameters with depth is interpreted as a decreased reliance on locomotory abilities for predator/prey interactions in the light-limited deep sea. The decline in metabolism with depth observed for pelagic cephalopods was significantly steeper than that previously observed for either pelagic fishes or crustaceans. We suggest that since strong locomotory abilities are not a priority in the deep sea, deeper-living cephalopods may rely more heavily on means of locomotion that are more efficient than jet propulsion via mantle contractions--means such as fin swimming or medusoid swimming utilizing the arms and extensive webbing present in many deep-living species. The greater efficiency of deeper-living cephalopods may be responsible for the observation that the decline in metabolic rates with depth is more pronounced for pelagic cephalopods than for fishes or crustaceans.

Inorganic N Assimilation and Ammonium Pools in a Deep-Sea Mussel Containing Methanotrophic Endosymbionts.

Undescribed mussels (seep mytilid 1a) harboring methanotrophic endosymbionts exhibit high biomass around hydrocarbon seeps on the Louisiana Slope of the Gulf of Mexico. These mussels assimilate ammonium and nitrate present at high concentrations in their environment. Pathways of assimilation were investigated by enzyme activity measurements and 15N tracer experiments. Glutamine synthetase was detected in all freshly collected mussels tested. Nitrate reductase activity was not always observed. Exposure to 15NH3 resulted in the appearance of millimolar concentrations of 15NH3 within the symbiont-containing tissues. The concentration of internal 15NH3 was several times higher than in the medium and correlated with 15NH3 assimilation rate. These results indicate that exogenous 15NH3 was taken up into a large internal pool before it was assimilated. Our results do not indicate the extent to which ammonium pools were within the host or symbiont or whether ammonium assimilation was facilitated by either partner exclusively. The observation of elevated internal ammonium concentrations is inconsistent with the "depletion-diffusion" mechanism of nutrient uptake proposed for algal-invertebrate symbioses and is suggestive of active ammonium uptake mechanisms across the host surface. Exposure to 15NO3- also resulted in the appearance of 15NH3, with internal 15NH3 concentration correlated with 15NO3- assimilation rate. This result indicates that 15NO3- was reduced more rapidly than it was assimilated and that 15NH3 derived from 15NO3- may also enter an internal ammonium pool. Assimilation of nitrate in the presence of millimolar concentrations of internal ammonium and reduction of 15NO3- in excess of assimilation is consistent with the functioning of dissimilatory nitrate reduction pathways with ammonium as a major endproduct. Such a mechanism may operate in other chemosynthetic symbioses that exhibit dissimilatory nitrate reduction.

Are there physiological and biochemical adaptations of metabolism in deep-sea animals?

From the earliest observations of deep-sea animals, it was obvious that they differed in many ways from shallower-living relatives. Over the years, there has been speculation that deep-sea animals have unusually low rates of biological activity; numerous adaptive scenarios explaining this have ben offered. However, these speculations and scenarios have rarely been tested due to the difficulty of data collection and the inevitable confounding of a number of major variables which covary with depth. In recent years, study of the metabolic properties of animals of several phyla from widely differing deep-sea habitats, including the hydrthermal vents, has made it possible, using comparative approaches, to test hypotheses concerning the metabolic adaptations of deep-sea animals.

Assimilation of inorganic nitrogen by marine invertebrates and their chemoautotrophic and methanotrophic symbionts.

Symbioses between marine invertebrates and their chemoautotrophic and methanotrophic symbionts are now known to exist in a variety of habitats where reduced chemical species are present. The utilization of chemical energy and reliance on C(1) compounds by these symbioses are well documented. Much less is known about their metabolism of nitrogen. Earlier work has shown that the tissues of organisms in these associations are depleted of N compared with those of other marine organisms, indicating that local sources of nitrogen are assimilated and that novel mechanisms of nitrogen metabolism may be involved. Although these symbioses have access to rich sources of ammonium (NH(4) and NH(3)) and/or nitrate, several investigators have proposed that N(2) fixation may account for some of these isotope values. Here we report that [N]ammonium and, to a lesser degree, [N]nitrate are assimilated into organic compounds by Solemya reidi, a gutless clam containing S-oxidizing bacteria, and seep mussel Ia, an undescribed mytilid containing methanotrophic bacteria. In contrast, Riftia pachyptila, the giant hydrothermal vent tube worm symbiotic with S-oxidizing bacteria, assimilated nitrate but not exogenous ammonium. The rates of assimilation of these sources are sufficient to at least partially support C(1) compound metabolism. N(2) assimilation was not exhibited by the symbionts tested.

Oxygen Consumption Rates and Metabolic Enzyme Activities of Oceanic California Medusae in Relation to Body Size and Habitat Depth.

Oxygen consumption rates were measured in 14 species of hydromedusae and 5 species of bathypelagic coronate scyphomedusae. Analysis of all individuals of all species of medusae showed the familiar pattern of decreasing specific oxygen consumption rate with increasing wet weight of animals. Citrate synthase (CS), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and pyruvate kinase (PK) activities were measured in more than 30 species of medusae. Octopine dehydrogenase, strombine dehydrogenase, and alanopine dehydrogenase were not detected in either hydromedusae or scyphomedusae. The allometric scaling phenomenon of decreasing activity in larger individuals was observed in Krebs cycle enzyme activities. LDH activities, on the other hand, increased with increasing wet weight. Most medusae were aerobically poised, with higher CS activities than LDH activities. However, several meso- and bathypelagic medusae, including the coronate scyphozoans Periphylla periphylla and Nausitho{e2dot} rubra, were anaerobically poised, possibly as a mechanism to assist in vertical migrations at low oxygen concentrations in the oxygen minimum layer. There is poor correlation between CS activities and oxygen consumption rates in these medusae when compared to previously investigated animals. To account for this poor correlation, we propose the hypothesis that medusan CS at the periphery of the maximum diffusion distance may be oxygen-limited and does not function at the normal in vivo rate. For pelagic medusae, there is no apparent decline in metabolic rate and metabolic potential, as determined by enzymatic activity, with increasing depth of occurrence, beyond the declines caused by the decrease in temperature with depth. These patterns are in contrast to the rapid declines in metabolic rates and metabolic potentials with depth that have been observed for pelagic fishes and crustaceans. Deep-living medusae have metabolic rates of a magnitude similar to those of bathypelagic fishes and crustaceans.

Homeoviscous Properties Implicated by the Interactive Effects of Pressure and Temperature on the Hydrothermal Vent Crab Bythograea thermydron.

Specimens of the hydrothermal vent crab Bythograea thermydron, collected from 13° N on the East Pacific Rise, were exposed to pressures greater than those in their natural habitat over a range of temperatures to assess how increased hydrostatic pressure affects a species that requires high pressure to survive. We measured heart beat frequency and contraction waveform at pressures ranging from 28 MPa (normal environmental pressure for this species) to 62 MPa at 5°, 10°, and 20°C. At 5°C, increased hydrostatic pressure induced bradycardia or acardia in conjunction with marked disruption of the ventricular contraction waveform. The animals did not survive following decompression. The effects of increased pressure were less pronounced at 10°C and almost negligible at 20°C. Our results support previous findings at subambient pressures which suggest that the lipid bilayers of cell and organelle membranes are the primary sites affected by short-term pressure variation in deep-sea organisms. We also found evidence of an adaptive mechanism of pressure temperature interaction in these animals from the eurythermic habitat of the hydrothermal vents.

Sulfide-Driven Autotrophic Balance in the Bacterial Symbiont-Containing Hydrothermal Vent Tubeworm, Riftia pachyptila Jones.

Hydrothermal vent tubeworms, Riftia pachyptila Jones, were maintained alive and studied on board ship using flow-through pressure aquaria. Simultaneous measurements of O2, ΣCO2, ΣH2S fluxes showed that the intact symbioses reach maximum rates of uptake of ΣCO2 (>2 µmole g-1 h-1) at about 90 µM ΣH2S. Measurements were made of hemolymph and coelomic fluid ΣCO2, ΣH2S, thiosulfate, pH, and hemoglobin concentrations in worms kept under various conditions of O2 and ΣH2S. Normal hemolymph pH appears to be about 7.5 and is not affected by ΣH2S and ΣCO2 concentrations within the ranges observed. We conclude that Riftia is specialized to provide sulfide to its symbionts with minimal interaction of sulfide with the animal metabolism. The uptake of sulfide is apparently by diffusion into the hemolymph, facilitated by the sulfide-binding properties of the hemoglobins. Both ΣCO2 and PCO2 are elevated in the hemolymph above their levels in the medium, although they are reduced under autotrophic conditions. Thus inorganic carbon is apparently concentrated from the medium into the hemolymph by an unknown mechanism.