Summary 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.

SUMMARY 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.

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.

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.

Calyptogena elongata is a small (about 7 cm maximum length) species of vesicomyid clam which lives at depths of 494–503 m, near the sill depth, in the Santa Barbara Channel in mildly reducing muds at low ambient oxygen concentrations. This species has abundant autotrophic sulphur- oxidizing bacteria in bacteriocytes in its gills. The stable carbon isotope composition values of its gills and other tissues range from −36 to −38 “, supporting the suggestion that the primary carbon source for this symbiosis is inorganic carbon fixed by the endosymbionts. This species of clam concentrates sulphide into its blood serum by using a sulphide-binding component and into the gills by using an unknown sulphide-binding activity. In both tissues, total H2S concentrations within the clam can greatly exceed those outside. This apparently enables the clam to concentrate sufficient sulphide from the mildly reducing muds to support the needs of its endosymbionts. Both of these binding activities are reversible in vivo as shown by the rapid declines in blood and gill sulphide levels when the clams are deprived of sulphide and the rapid concentration of sulphide into the blood and gills when it is provided. For example, within minutes of exposure to 65 micromolar H2S, gill and blood total H2S concentrations in individual C. elongata exceed the external concentration; within 2 h they reach maximum concentrations of about 2 mmol l-1. When such experiments are carried out under anoxic conditions, the blood and gill total H2S concentrations approach saturation (10–20 mmol l-1), indicating that under oxic conditions the oxidation of sulphide by the clam and its endosymbionts holds the binding components below saturation and enables them to protect the animal tissues and endosymbionts from toxic concentrations of sulphide. In contrast to these results for C. elongata, our experiments show that the host of another chemoautotrophic symbiosis, Solemya reidi, does not concentrate sulphide from the medium into either its blood or its gills. Data are presented which indicate that the serum sulphide-binding component is a large molecular mass molecule with Zn2+ at the active site. This study strongly supports the model of vesicomyid functioning in which the blood- borne sulphide-binding component concentrates sulphide from the reducing environment around the clam's foot and transports this sulphide to the symbionts in the gills. Data are also presented which indicate that the clam oxidizes some sulphide to thiosulphate and transports this to the gills as well. Thus, individual C. elongata, like the previously studied C. magnifica, appear to bridge the reducing and oxidizing zones of their habitats to provide needed substrates to their endosymbionts. Examination of these two species and the anatomy of other vesicomyid species suggest that vesicomyid clams are functionally quite conservative.

Present address and address for correspondence: Bamfield Marine Station, Bamfield, B.C. V0R 1B0, Canada. The effects of temperature and pH on haemocyanin oxygen-binding were compared for three species of diurnally vertically migrating and two species of non-migrating, pelagic oplophorid shrimps from the deep sea off the Hawaiian island of Oahu. The effects of L -lactate were also measured for three of these species. Haemocyanin concentrations were higher in the haemolymphs of oplophorids that migrate vertically (39.4, 46.8 and 57.6 mg ml −1 ) than in those of non-migrators (26.0 and 36.4 mg ml −1 ). Moderately high Bohr effects were found for vertically migrating and non-migrating oplophorids at all temperatures examined (5–25°C,φ=−0.46 to −0.80, and −0.55 to −0.88, respectively). The vertically migrating species had temperature-sensitive haemocyanins (Δ H =−23.1 to −41.2 kJ mol −1 ) across the normal temperature range (5–25°C) encountered during diurnal vertical migration. This results in haemocyanins that have relatively high affinities ( P 50 =0.80-1.06 kPa at pH 7.8,5°C) at the low temperatures and low O 2 partial pressures (approximately 2.66 kPa O 2 at 5°C) found at depth, and low affinities ( P 50 =4.00-4.66 kPa at pH 7.5, 25°C) at the higher temperatures and higher O 2 partial pressures (approximately 13.33-17.50 kPa at 25°C) found in the near-surface waters. In contrast, the non-migrating species, which live within a narrower temperature range (3–6°C) and at a constant, low partial pressure of O 2 (2.66-4.00 kPa), have haemocyanins with a high affinity for oxygen ( P 50 =0.67-0.93 kPa at pH 7.8, 5°C) and lower sensitivity to temperature (Δ H =−4.2 to −21.6 kJ mol −1 1). The effects of temperature on the haemocyanin oxygen-affinities of the vertical migrators appear to be highly adaptive, enabling these haemocyanins to be functional across the entire depth (and thus, temperature and oxygen partial pressure) range encountered.

Pelagic fishes with an ability to swim in strong bursts have previously been shown to have large size-dependent increases (positive aUometric scaling exponents) in the activities of glycolytic enzymes in white skeletal muscle. This scaling of glycolytic activity has been hypothesized to provide the anaerobic power upporting the size-independence of relative burst swimming speeds (body lengths s −1 ) in these fishes. This paper presents tests of several predictions of this hypothesis, using different-sized individuals of two pelagic teleosts, the kelp bass (Paralabrax clathratus) and the freshwater rainbow trout ( Salmo gairdneri ), and a flatfish, the Dover sole ( Microstomus pacificus ). In the two pelagic species, an increase in body size was accompanied by an increase in activities in white muscle (i.u.g wet mass ofmuscle −1 ) of lactate dehydrogenase (LDH), an indicator of potential for anaerobic glycolysis, and creatine phosphokinase (CPK), an enzyme that helps maintain stable ATP concentration during muscular activity. Activities of citrate synthase (CS), an indicator of the potential for aerobic metabolism, decreased with size. In the flatfish, activities of all enzymes in white muscle decreased with body size, a trend proposed to reflect lack of adaptive value of strong burst swimming ability in this benthic fish. Activities of LDH and CS were size-independent in brain of flatfish, indicating that the scaling patterns observed in the muscle of this species were related to muscle function, not to common, organism-wide changes with size. In white muscle of P. clathratus , total protein and soluble protein concentrations and buffering capacity increased with body size in parallel, but myofibrillar protein was size-independent. These results suggest that the capacity for anaerobically powered work and the maximal potential to generate force scale only modestly in relation to total body mass and therefore do not appear to be functionally related to the pattern of glycolytic scaling. Thus, these data support the hypothesis that the functional role of the strongly positive scaling of glycolytic enzymes in the white muscle of pelagic fish is to provide increased power during burst swimming in larger-sized fishes.

Unlike most pelagic crustaceans, the deep-sea shrimp Notostomus gibbosus (A. Milne-Edwards) (Oplophoridae) is positively buoyant, possessing a dorsally enlarged carapace which contains a low-density fluid. This fluid comprises 43% of the animal's wet mass, has a low pH, and gives a lift of 17.7 mg ml −1 ; when this fluid is drained, the animal sinks. Low density is achieved by the replacement of less buoyant ions with ions which reduce density via two mechanisms: a change in total solute mass by the use of ions of lesser mass, and an ion-specific disruption of the structure of water molecules (resulting in an increase in fluid volume) caused by ions having large, positive partial molal volumes. The presence of large amounts of trimethylamine (Me 3 NH + ), a relatively large, heavy ion which, together with NH 4 + , replaces nearly 90 % of the Na + in the carapace fluid, results in little change in the total solute mass of the carapace fluid of N. gibbosus (33.2‰) relative to sea water (approximately 34.1‰). Reduced fluid density is primarily a result of the large, positive partial molal volumes of Me 3 NH + and NH 4 + , rather than a function of reduced solute masses.

Solemva reidi Bernard is a gutless clam that lives in burrows in reducing sediments, and harbours intracellular sulphur-oxidizing bacteria in its gills. Clams were incubated in various concentrations of sulphide and thiosulphate for up to 65 h in a flow-through respirometer. Fluxes were determined by continuous sampling of the respiratory medium with analysis of CO 2 , O 2 and sulphide by gas chromatography and analysis of thiosulphate, sulphite (and sulphide) by HPLC using monobromobimane-denvatized discrete samples. Net CO 2 uptake was shown to occur with exposure to 50–100μmol1 −1 sulphide and greater than 225μmoll −1 thiosulphate; sulphide oxidation and thiosulphate uptake were also demonstrated. 45 CaCO 3 deposition in the shells of. S. reidi was found to be insignificant compared to the net CO 2 flux measured in the presence of low levels of sulphide. In experiments conducted under various O 2 conditions, O 2 limitation, produced by a combination of low [O 2 ] and low water flow, was shown to inhibit sulphide oxidation and to prevent Co 2 uptake. However, if O 2 supply was not limited by low flow rates, in the presence of low [O 2 ] (25–40 μmoll −1 ) S. reidi showed rates of O 2 and sulphide consumption and CO 2 uptake near the maximum levels determined under high [O 2 ] conditions, indicating the potential for net Co 2 uptake in the low [O 2 ] conditions presumed to exist in the animal's burrows. Thiosulphate levels in the blood of S. reidi were analysed and shown to increase rapidly during incubation in sulphide. These levels reached an apparent steady state (approx. 300μmoll −1 ) in recently captured clams after 1 h of incubation. However, both O 2 limitation and time in captivity (>43 days after capture) caused a marked increase in the blood thiosulphate levels, which exceeded 2.5mmoll −1 after 16 h of exposure to sulphide. These results indicate that blood thiosulphate is transported to the bacteria and further oxidized, and that sulphide and thiosulphate oxidation are oxygen-dependent. In analyses of gill tissues for elemental sulphur, we found a wide range in the levels of sulphur stores. Calculations indicated these to be a small fraction of the total flux of sulphur maintained during continuous sulphide oxidation. Estimates of Co 2 :o 2 : sulphide ratios suggest CO 2 fixation efficiencies similar to those of chemolithoautotrophic bacteria. Assuming translocation and oxidation of symbiont-fixed organic compounds, the net uptake of CO 2 by S. reidi in the presence of reduced sulphur compounds suggests that this intact symbiosis may be able to meet its organic carbon needs through autotrophy.