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Home , Riftia pachyptila, Trophosome

l MARINE ECOLOGY PROGRESS SERIES Vol. 174: 151-158,1998 Published November 26 Mar Ecol Prog Ser

Metabolic responses of the to severe

Cordelia ~rndt',~.*,Doris Schiedek2, Horst Felbeckl

'University of California San Diego, Scripps Institution of Oceanography. La Jolla. California 92093-0202. USA '~alticSea Research Institute at the University of Rostock, Seestrasse 15. D-181 19 Rostock-Warnemuende. Germany

ABSTRACT: The metabolic capabilit~esof the hydrothermal vent tube worm Riftia pachyptila to toler- ate short- and long-term exposure to hypoxia were investigated After incubating specimens under anaerobic conditions the metabolic changes in body fluids and tissues were analyzed over time. The tube worms tolerated anoxic exposure up to 60 h. Prior to hypoxia the dicarboxylic acid, malate, was found in unusually high concentrations in the (up to 26 mM) and tissues (up to 5 pm01 g-' fresh wt). During hypoxia, most of the malate was degraded very quickly, while large quantities of succinate accumulated (blood: about 17 mM; tissues: about 13 pm01 g-l fresh wt). Volatile, short-chain fatty acids were apparently not excreted under these conditions. The storage compound, , was mainly found in the and appears to be utilized only during extended anaerobiosis. The succinate formed during hypoxia does not account for the use of malate and glycogen, which possibly indicates the presence of yet unidentified metabolic end products. Glutamate concentration in the trophosome decreased markedly durlng hypoxia, presumably due to a reduction in the autotrophic function of the symb~ontsduring hypoxia. In conclusion, R. pachyptila is phys~ologically well adapted to the fluctuations freq.uently occurring In the vent habitat.

KEY WORDS: Riftia pachyptda - Anaerobic metabolism . Hypoxla . Hydrothermal vents

INTRODUCTION indicate that these mainly respire aerobically to gain energy from degrading organic associated with hydrothermal vents is mainly food stuff (Hand & Somero 1983). While other animals limited to the narrow region where the hot anoxic vent can escape from unfavorable conditions, many vent effluent mixes with the oxygenated bottom cannot do so. The vestimentiferan worm Riftia water (Corliss et al. 1979, Edmond et al. 1982, Jan- pachyptila, for example, is a sessile tube-dwelling nasch & Mottl 1985). The environmental conditions in species that has only a limited ability to respond be- the vent area are extremely unstable and change haviorally to unfavorable changes in its environment. rapidly. Johnson et al. (1988a) demonstrated that ani- Juvenile tube worms, in particular, mlght be affected mals living in the vent environment may experience by hypoxic conditions since they frequently settle at high concentrations of as well as extreme the bottom part of the tubes of adult specimen and are, hypoxia, depending on the vent water flow. Significant therefore, more exposed to venting waters (authors' changes in the oxygen concentration could be detected pers. obs.). around these organisms within a period of seconds as Riftia pachyptila is, because of its enormous size of well as over longer periods (Johnson et al. 1988a, h). up to 1.5 m length (Jones 1981a), one of the most con- This extreme variation in oxygen availability is likely spicuous members of the vent community which forms to have an important impact on the energy metabolism dense clusters around the hypoxic vent effluents. Adult of vent animals that, in the long run, must live aerobi- specimens have no digestive system and derive the cally. activities found in the tissues of vent major part of their nutrition from intracellular sulfide- oxidizing housed in the trophosome tissue (Jones 1981b). The chemoautotrophic syrnbionts require

O Inter-Research 1998 Resale of full article not permitted 152 Mar Ecol Prog Ser

access both to sulfide and to an oxidant in order for flow-through aquarium system similar to that de- to take place. In addition to oxygen, scribed in Goffredi et al. (1997). To maintain the speci- the bacteria are able to respire and nitrite mens in a healthy condition, the flow-through aquaria (Hentschel & Felbeck 1993). This metabolic pathway were kept at 15°C and supplied with sea water con- could be a great advantage for the symbionts during taining about 2 mM , 300 FIM sulfide, environmental hypoxia. While oxygen might already 120 yM oxygen and 0.4 mM nitrogen ('maintenance be depleted in the habitat because of its rapid reaction condition'). The pH of the water was kept at pH 6.5 by with sulfidic vent waters, nitrate could still be available an automatic pH-controller that pumped HC1 into the due to its lower reactivity with sulfide (Jannasch & mixing column. The concentration of the gases and Mottl 1985). However, R. pachyptila itself cannot take sulfide were periodically monitored by gas chromato- advantage of this pathway since nitrate respiration is graphy (Childress et al. 1984). restricted to and has not been found Experimental procedure. The tube worms were in the or any other metazoan . kept for at least 12 h under maintenance conditions The various responses of to environ- before an experiment started. To investigate responses mental hypoxia have been rev~ewed by different to severe hypoxia, the conditions in the high pressure authors (Bryant 1991, Grieshaber et al. 1994). A com- aquaria were changed by bubbling nitrogen gas mon response to anoxia is a switch to fermentative instead of oxygen through the mixing column, which pathways in addition to a reduction in metabolic allowed the oxygen concentration to be maintained energy demands. In contrast to aerobic respiration, between 0 and 6 yM. The total CO2 and sulfide con- which provides energy via the respiratory chain, centrations were maintained as usual.. Specimens were metabolism during anoxia must provide energy by incubated for different periods of time (0, 6, l?, 48 or means of substrate-linked phosphorylation. Some 60 h) under hypoxic conditions. After 60 h, worms were species produce lactate and opines from the degrada- still considered as healthy based on their response to tion of carbohydrate, which is the most important external stimuli and the red color of their plumes, energy source during hypoxia. Other anaerobic path- which turn grayish-slimy in dying specimens. ways include the fermentative generation of succinate To investigate whether Riftia pachyptila excretes and volatile, short-chain fatty aclds. Even with a rela- volatile, short-chain fatty acids, 3 specimens were tively high energy gain, free-living animals can utilize incubated in hypoxic sea water in heat-sealed poly- anaerobiosis only for a restricted time due to their ethylene bags (about 250 pm thick) for 12 h. At the end dependence on limited internal glycogen stores. of the experiment, the incubation water from the bags Previous investigations have suggested a substantial was sampled, adjusted to an alkaline pH and stored tolerance of Riftja pachyptila to hypoxia, as the worm frozen. The fatty acids were later removed by steam was shown to survive at least 36 h without oxygen distillation according to Kluytmans et al. (1975) and (Childress et al. 1984).However, the underlying meta- their concentrations determined with a Hewlett bolic mechanisms which enable R. pachyptila to toler- Packard gas chromatograph equipped with a flame ate periods without oxygen have not been addressed. ionization detector. The column used was a 6 ft (ca In the present study we investigated the metabolic 1.8 m) long glass column (Supelco) filled with 10% SP pathways of R. pachyptila that sustain activity in the 1200 and 1 % H3PO4 on 80/100 Chromosorb WAW absence of oxygen. The questions we asked in our At the end of an incubation period, the worms were work were: first, which mechan~sms are used by R. removed from the experimental chamber and immedi- pachyptila to survive extended periods of anoxia; and, ately dissected. Blood was collected from the main second, is the autotrophic functioning of the symbionts ventral and dorsal vessel as well as from the coelomic of the tube worm affected in any way by oxygen cavity. Plume, body wall, vcstimentum, and tropho- deficiency? some were rinsed, blotted dry and freeze-clamped in liquid nitrogen. All samples were stored at -80°C before extraction. For the immediate analysis on MATERIALS AND METHODS board, the tissues were weighed on a motion compen- sated ship-board balance system (Childress & Mickel Animal collection. Specimens were collected during 1980). the 'HOT96' expedition to the hydrothermal vents at Enzyme activities and metabolite determination. 9" N and 13"N along the East Pacific Rise (2600 m The activities of lactate dehydrogenase, octopine depth). The animals were recovered with the sub- dehydrogenase, alanopine dehydrogenase, and strom- mersible 'Nautile' of the NO 'Nadr' (IFREMER, France) bine dehydrogenase were determined spectrophoto- in a thermally insulated container. After reaching the metrically in crude homogenates of plume, vestimen- surface, the worms were repressurized to 210 atm in a tum, trophosome, and body wall tissue of freshly Arndt et al.: Anaerobic metabolism in Rlftiapachyptila 153

caught worms according to Schiedek (1997). The assay temperature was chosen as 25°C in order to be compara- ble to most other published data. To analyse the concentrations of metabolites indicative of anaerobiosis in the tissues and body fluids of Riftja pachyptila, neutralized perchloric acid extracts were prepared according to Schiedek & Schoettler (1990). The ex- 0 6 17 48 60 0 6 17 48 60 traction and of glycogen period of anaerobiosis [h] period of anaerobiosis [h] was performed according to Schoettler ventral blood dorsal blood vcslimcn~um body wall (1978). Standard enzymatic methods coloemic fluid Irophosomc plume were for measuring Fig. 1. Riftiapachyptlla. Succinate concentrations in (a) body fluids and (b) tis- malatel and D- and =-lactate (Berg- sues after exposure to anaerobic conditions. Bars represent the mean values + meyer 1985). succinate was deter- SD.n = 3 (0 h), 4 (6 h), 5 (l? h), 9 (48 h), 4 (60 h). When no bars are shown, no mined according to Michal et al. samples were available. The concentrations in the body fluids, body wall, and (1976). The separation and detection of vestimentum increased significantly during the first 6 h (p c: 0.05) free amino acids was performed via HPLC after Schiedek (1997). To detect changes of the metabolite concentrations No accumulation of volatile fatty acids was found in during the incubations, the results of different incuba- the incubation water of Riftia pachyptila.None of the tion periods were compared to the initial values using free amino acids tested in the tissues and body fluids of a 2-tailed I-test for unpaired results. If p was smaller R. pachyptila increased significantly during hypoxia. than 0.05, the change was considered significant. Compared to other amino acids, alanine concentration was very high in the body wall tissue and had a far lower level in other tissues (Table 1). However, no sig- RESULTS nificant hypoxia-related changes could be detected.

Accumulation of anaerobic metabolites Degradation of storage compounds The dicarboxylic acid succinate was found in very during severe hypoxia high concentrations in the blood (about 7 mM) and tissues of Riftia pachyptila (3 to 10 pm01 g-l fresh wt) Malate concentrations decreased rapidly in the even under aerobic conditions. However, exposure to blood and tissues during oxygen deficiency (Fig. 2a). severe hypoxia raised the succinate levels in all body Under aerobic maintenance conditions up to 26 mM compartments of the tube worm. After 6 h without malate could be detected in the blood. After 6 h of oxygen, the succinate level in the body fluids in- anaerobic exposure, malate concentration was reduced creased by about 17 mM (Fig. la). No significant fur- to about 2 mM in the vascular blood as well as in the ther accumulation could be detected during longer anoxic incubation pen- Table 1. Riftiapachyptila. Changes In concentration of the amino acids aspartate ads. The succinate concentration was and alanine durinq anaeroblosis In different tissues. Numbers indicate mean in the same range in the ventral and values i standard deviation. Values In parentheses are numbers of specimens dorsal blood and in the coelomic fluid. analysed. n = 4 for all 6 and 48 h ~ncubations.nd = not determined Succinate accumulated in the tissues throughout the anoxic incubation time Amino acid Tissue Time of anaerobic exposure (h) (Fig lb), increasing in both the body (pm01 g-' fresh I&) 0 6 48 wall and the vestimentum from about Aspartate Plume 3 pm01 g-' fresh wt to 16 pm01 g-' Vestimentum fresh wt within 60 h. Under hypoxia, Body wall the trophosome showed no significant Trophosome changes in the succinate concentra- Alanine Plume Vestin~entum tion, while succinate in the plume tis- Body wall sue increased by about 4 pm01 g-' Trophosome fresh wt from 6 to 48 h. Mar Ecol Prog Ser 174: 151-158, 1998

utilization of glycogen in both the body wall and vestimentum was much less than in the trophosome even after 60 h of hypoxia, when it decreased signifi- cantly by about 17 and 14 pm01 glyco- syl units g-' fresh wt, respectively. The glycogen concentration of the plume tissue was very low and decreased only minimally during hypoxia. 0 6 17 48 60 0 6 17 48 60 Aspartate, which was present in the periodof anaerobiosis[h] periodof anaerobiosis[h] tissues of Riftiapachyptila in very low ventral blood dorsal blood vestirnentum body wall concentrations (Table l), was com- coloemicfluid trophosorne plume pletely depleted within 6 h of anaero- Fig. 2. Riftia pachyptila. Malate concentrations in (a) body fluids and (b) tissues bic exposure. Glutamate was found in after exposure to anaerobic conditions. Bars represent the mean values 2 SD. high quantities in the trophosome tis- n = 3 (0 h), 4 (6 h), 5 (If h), 9 (48h), 4 (60 h). When no bars are shown, no samples sue (Fig. 4).Its concentration declined were available. The concentrations in the body fluids, body wall, and vesti- continuously over the anaerobic incu- mentum decreased significantly during the first 6 h (p i 0.05) bation period, showing a significant reduction after 48 h. coelomic fluid. In the tissues of Riftia pachyptila, the malate level was highest in the vestimentum (about 4 ~.lmolg-l fresh wt), while the other tissues had lower Enzyme activities concentrations (Fig. 2b). A significant reduction in malate content could be found after 6 h of anaerobic Octopine dehydrogenase, alanopine dehydrogenase exposure in the body wall and vestimentum of R. and strombine dehydrogenase activities were not pachyptila. found in any of the samples. Lactate dehydrogenase Glycogen was abundant in the trophosome tissue could be detected in moderate activities both in vesti- (about 100 pm01 glycosyl units g-l fresh wt), but less mentum and body wall (4 pm01 g-l fresh wt min.' and concentrated in the remaining body tissues, which 3 pm01 g-' fresh wt min-l, respectively). contained 17 to 35 pm01 glycosyl units g-' fresh wt (Fig. 3). Within the first 17 h of hypoxia, a significant degradation of glycogen could not be detected in any DISCUSSION of the tissues of the tube worm. After 48 h, glycogen store in the trophosome tissue had decreased signifi- When incubating Riftia pachyptila for different peri- cantly by about 60 pm01 glycosyl units g-' fresh wt. The ods of severe hypoxia, the tube worms were found in good shape even after 60 h of anaerobic exposure. All specimens were still actively responding to tactile stimuli and the1.r plumes had a fresh red color as is

0 6 17 48 60 periodof anaerobiosis[h] vestirnentum H bodywall trophosome plume periodof anaerobiosis[h] Fig. 3. Riftia pachyptila. Glycogen concentrations in tropho- some, body wall, plume, and vestimentum after exposure to Fig. 4. Wtia pachyptila. Glutamate concentrations in the anaerobic conditions. Bars represent the mean values * SD trophosome after exposure to anaerobic conditions. The mean n = 3 (0 h),4 (6 h), 5 (17 h], 9 (48 h),4 (60 h).When no bars are values i SD are plotted. n = 3 (0 h), 4 (6 h), 5 (17 h),9 (48 h), shown, no samples were available. The concentration in the 4 (60 h). The concentration decreased sigIzlf~cantlyafter 48 h trophosome decreased significantly after 48 h (p c 0.05) (p 0.05) Arndt et al.: Anaerobic me1tabolism In Rirtia pachyptila 155

typical of healthy specimens. However, after 60 h of of free oxygen inside the tissues of R. pachyptila hypoxia, in contrast to shorter incubation periods, the (Fisher et al. 1989, Hentschel & Felbeck 1993). In addi- plumes of all specimen were retracted into their tubes tion to hypoxic conditions, the elevated CO2-concen- in the incubation vessels. Since severely stressed R. trations in the blood of the tube worm (Childress et al. pachyptila are often found with plumes withdrawn into 1993) may affect the succinate concentration. This their tubes, this could indicate that the health of the metabolic response has been discussed for parasitic specimens had begun to deteriorate due to their hav- helminths which form succinate under aerobic condi- ing reached maximal anaerobic tolerance. tions, even though they are able to gain energy aerobi- An analysis of different body compartments of Riftia cally (Barrett 1976). It is believed that the production pachyptila suggests a significant role of succinate in of organic acids may avoid the problem of tissue acidi- the anaerobic metabolism of the tube worm. This fication which is caused by the high CO, levels result is not surprising considering the physiological frequently found in the tissues of the terrestrial host responses of other experiencing animals of helminths (Podesta et al. 1976). A third anoxia. For most species investigated, explanation for the high succinate levels could be that succinate accumulation in the body fluids and tissues is the chemoautotrophic bacteria of R. pachyptila excrete a characteristic feature of their anaerobic metabolic succinate as a metabolic product (Felbeck & Jarchow response. Succinate usually originates from aspartate 1998) which is then transferred to the host. in early stages of anoxia, while glycogen is utilized The large and rapid accun~ulationof succinate under during extended anaerobiosis to generate energy hypoxic conditions demonstrates the importance of (, ATP) via substrate phospho- this fermentative process for Riftia pachyptila (Fig. 1). rylation (Bryant 1991). Under anaerobic conditions, succinate formation from Succinate metabolism in Riftia pachyptila appears to glucose results in a larger energy yield compared to differ from that of other animals. Unusually high con- lactate fermentation or other dehydrogenase reactions centrations of succinate were found in the body fluids such as opine formation. Instead of 2 m01 ATP per gly- and tissues of the tube worm even under aerobic con- cosy1 residue, twice as much is generated by succinate ditions (Fig.l), whereas other terrestrial, fresh water, production. The metabolic source of the extremely and marine species examined display succinate con- high succinate content after hypoxlc exposure of R. centrations below 0.1 pm01 g-' fresh wt in their tissues pachyptila (body fluids: up to 46 mM; tissues: up to (Grieshaber et al. 1994). The normal level found in the 23 pm01 g-l fresh wt) is unknown. Glycogen, a com- tissues of R. pachyptila was 30 to 50 times higher (up to mon storage compound, was most concentrated in the 5 pm01 g-' fresh wt). Succinate is even more concen- trophosome tissue, reaching values of up to 157 pm01 trated in the body fluids of the tube worm. Compared glycosyl units g-' fresh wt (Fig. 3). Its depletion, al- to the level of 0.1 mM in the coelomic fluid of the lug- though demonstrable, cannot account for the produc- worm Arenicola marina (Schoettler & Bennett 1991), tion of succinate during the initial phases of anaerobic the succinate concentrations in this compartment and exposure, because a significant degradation of glyco- in the vascular blood of R. pachyptila are about 70 gen could only be detected after 48 h. Therefore, a times higher. further carbon source for the generation of succinate The elevated succinate level in the tube worm might must be available. suggest that the function of succinate is that of a CO2- Aspartate is degraded during early anaerobiosis in carrier. Felbeck (1985) showed that C02from the envi- many marine animals. Its breakdown is responsible for ronment is incorporated into the C-4 organic acids the accumulation of succinate during early stages of malate and succinate after entering the blood via the anoxia and results in an increase of the alanine con- plume. The author states that succinate is mainly deliv- centration due to a transamination reaction (Zebe ered to the chemoautotrophic bacteria in the tropho- 1975, Collicutt & Hochachka 1977, Felbeck 1980). some, where it can be decarboxylated after conversion However, since the concentration of aspartate is very to malate. However, it may also be directly utilized by low in Riftia pachyptila, it appears to play only a other tissues of Riftia pachyptila. An alternative expla- negligible role in the formation of succinate. Only nation for the high succinate levels in the tissues might about 2 pm01 g-' fresh wt could be measured in both be that the tube worms rely partially on anaerobic the body wall and vestimentum under aerobic condi- metabolic processes even under aeroblc conditions. tions (Table 1). Even if these amounts are depleted The unusual oxygen binding properties of the blood of after 6 h of anoxia and, possibly, metabolized to succi- R. pachyptila (Arp & Childress 1981) would seem to nate and alanine, they cannot account for the extent of support this possibility. Both the vascular and the succinate production. coelomic have extremely high affinities The analysis of the malate concentrations provides for oxygen, which could cause very low concentrations insight into the anaerobic succinate generation in Riftia 156 Mar Ecol Prog Ser 174: 151- 158, 1998

pachyptila. Malate, formed by glycogen depletion, is sulfide oxidation by nitrate is lower than via oxygen known as an important intermediate in the anaerobic (Stewart 1988), the bacteria could produce only rela- metabolism of invertebrates, serving as a substrate for tively little ATP and, therefore, could incorporate less mitochondrial energy conversion (Bryant 1991). Under COz. An indication for the declining autotrophic func- hypoxia, it is oxidized (clockwise) as well as reduced tion of the bacteria may be the decrease of free gluta- (counterclockwise) in the rnitochondrial tricarboxylic mate in the trophosome tissue of R. pachyptila under acid cycle and leads to the formation of succinate and anoxic conditions (Fig. 4). Glutamate was identified as the short-chaln fatty acids acetate and propionate. Pre- one of the bacterial metabolic products excreted after vious investigations on R. pachyptrla showed high con- CO2-fixation (Felbeck & Jarchow in press). Thus, a centrations of malate (about 5 mM) in the coelomic decline of its concentration during anoxia could pos- fluid and the blood of freshly recovered tube worms sibly be caused by a reduced generation due to the (Felbeck unpubl. data). Our data display even higher limited CO2-supply. malate concentrations of up to 26 mM in the body While malate appears to be the major anaerobic sub- fluids of worms exposed to aerobic maintenance con- strate in Riftia pachyptila during early anaerobiosis, ditions for at least 12 h. To our knowledge, these most of the glycogen is used only during extended numbers are among the highest ever detected in any anoxia. The extent of energy reserve mobilization is invertebrate species. The tissues of R. pachyptila also more evident if one calculates the sum of substrates display elevated malate concentrations of up to 5 pm01 mobilized during anoxia in all body compartments of g-' fresh wt. the tube worm and compares it to the sum of anaerobic Previous investigations on have given some products accumulating (Table 2). A specimen of R. insight concerning the metabolism of malate during pachyptila of 100 g fresh wt would degrade about anoxia. Relatively high concentrations were found in 1400 pm01 substrate during the first 6 h of anoxia, the blood and tissues of Lumbriculus variegatus and while an additional 1600 pm01 is used up to the end Hirudomedicinalis (Zebe et al. 1981, Putzer 1985). of the 48 h incubation period. The anaerobic products Malate was almost completely catabolized in the first identified in this study accumulating in the same indi- hours of anoxia in both species, while similar quanti- vidual add up to only 900 pm01 after 6 h and up to only ties of succinate accumulated. Our data suggest a an additional 400 pm01 within the following 42 h of similar metabolic mechanism in Riftia pachyptila. experimental anoxia. It is, therefore, likely that still Malate, presumably formed in the plume tissue as a unidentified anaerobic end products result from the CO2-carrier (Felbeck 1985, Felbeck & Turner 1995), long-term degradation of glycogen. might also serve as an energy source for anaerobic While many marine invertebrates display high metabolic processes. Under aerobic conditions, malate enzyme activities responsible for the reaction of pyru- is decarboxylated in the trophosome tissue to supply vate with free tissue amino acids in order to produce the chemoautotrophic bacteria with CO2 (in addition to CO2 dissolved in the blood). Under these circum- Table 2. Riftia pachyptila. Molar balance of substrates and stances, the bacterial sulfide oxidation generates products during anaerobic metabolism calculated for a 100 g enough energy to support autotrophic CO2-fixation. specimen. Numbers represent the sum of identified anaerobic During hypoxia, in contrast, the bacterial energy pro- substrates and products utilized or accumulated after 6 and 48 h of anoxla In all body compartments of the tubeworm. The duction and, therefore, the autotrophic function of the weight distribulion among the individual tissues was: body symbionts, appears to be restricted. This makes it fluids, about 40% (Felbeck pers. obs.);plume, 16 * 3%; vesti- likely that the symbionts fix CO2 less readily under menturn, 14 * 2%; body wall, 10 + 2%; trophosome, 15 & 2% anaerobic conditions than they do under aerobic con- (n = 9 for the latter numbers). The utilization of glycogen is ditions. Indeed, a decrease of CO2-uptake in intact R. calculated for 0.5 glycosyl units pachyptila under anaerobic conditions was observed (Childress pers. comm.) Since the malate stores cannot Exposure Anaeroblc sub- Anaerobic end- to anaero- strates (pmol) products (pmol) be rebuilt under these conditions, this might be a cause biosis (h) for the rapid and almost complete utilisation of these reserves. 6 Malate 640 Succinate 900 The only alternative to the oxidation of sulfide with Aspartate 50 oxygen would be the use of nitrate as an oxidant. As 0.5 glycosyl units 710 Z 1400 1900 the results of Pospesel et al. (in press) show, nitrate concentrations in the blood of Riftia pachyptila may be Malate 710 Succinate 1300 Aspartate 50 up to 100 times the level in ambient sea water. Nitrite, 0.5 glycosyl un~ts 2240 a product of nitrate respiration, was also conspicuously 3000 elevated. However, because the energy output from Arndt et al.: Anaerobic metabohsm in R~ftiapachyptila 157

opines, no alanopine, strombine or octopine dehydro- supply of organic carbon, it appears doubtful whether genase could be detected in the tissues of Riftia any metabolic products still capable of producing pachyptila. We could detect low activities of lactate energy are excreted into the environment. Instead, dehydrogenase in body wall and vestimentum of R. they are probably recycled internally using the meta- pachyptila (about 4 and 3 pm01 g-' fresh wt min-l, bolic capabilities of the bacteria. respectively). Lactate dehydrogenase activities in the trophosome were also found by Hand & Somero (1983). Acknowledgements. This research was supported by the However, only traces of L-lactate (<0.1 pm01 g-' fresh National Science Foundation grant OCE 93-14525 to H.F., a European Union grant (TMR-LSF: MAST3-CT95-0040) to wt) and no D-lactate could be measured in any body C.A. and a BMBF grant (DYSMON 03F0123E) to D.S. and compartments of the tube worm even after 48 h of C.A LVe thank the captains and the crew of the RV 'Wecoma' anaerobic exposure. (USA), NO 'Nadir' and the submersible 'Nautile' (IFREMER, Other fermentatlon products known to be accumu- France). Special thanks are due to Ute Hentschel for helpful comments and critical reading of the manuscript. lated and excreted in marine invertebrates under anaerobic conditions are volatile fatty acids such as propionate and acetate. We were not able to detect these compounds in the anaerobic incubation water of LITERATURE ClTED Riftia pachyptila. However, we cannot exclude their Arp AJ, Childress JJ (1981) Blood function in the hydro- production. It was not possible to test whether the thermal vent tube worm. Science 213:342-344 matellal of the incubation bags (about 250 pm thick Barrett J (1976) Bioenergetics In helrninths. In: van den polyethylene) was completely impermeable to short- Bosche H (ed) Biochen~istryof parasites and host-parasite chain fatty acids. Technical data were not available relationships. Elsevier, Amsterdam, p 67-77 Bergmeyer U (1985) Methods of enzymatic analysis, 3rd edn. from the literature. Any permeability would have re- Vol VII. VCH, Weinheim sulted in a partial loss of fatty acids from the incubation Bryant C (1991) Metazoan life without oxygen. Chapman and bags. Further experiments are necessary before a Hall. London definitive statement about the significance of this Childress JJ, Arp AJ, Fisher CR Jr (1984) Metabolic and blood characteristics of the hydrothermal vent tube worm Riftia anaerobic end product of R. pachyptila can be made. pach~,ptila.Mar Biol 83:109-124 Other adaptations to short-term anoxia were found Childrcss JJ, Lee RW, Sanders NK, Felbeck H, Oros D, Toul- by Childress et al. (1984), who reported oxygen con- mond A, Desbruyeres D,Kennicutt MC 11,Brooks J (1993) sumption rates in Riftia pachyptila regulated down to Inorganic carbon uptake in hydrothermal vent tube low oxygen partial pressures (p02).The authors calcu- worms facilitated by high environmental pC02. Nature 362:147-149 lated that the tube worm must be able to maintain its Childress JJ. Mickel TJ (1980) A motion compensated ship- aerobic metabolism under anoxia for about 0.56 h if board precision balance system. Deep-Sea Res 27A: their oxygen stores in the blood are saturated. These 965-970 metabolic properties may help R. pachyptila to sustain Collicutt JM, Hochachka PW (1977) The anaerobic oyster heal-t: coupling of glucose and aspartate fermentat~on. aerobic functioning during periods of withdrawal into J Comp Physiol 115:147-157 the tube, which is the only way to protect themselves Corliss JB, Dymond J, Gordon LI, Edmond JM, Herzen RPV, from predators. Whether high-energy phosphagens Ballard RD. Green K, Williams D, Ba~nbridgeA, Crane K, are also involved in a fast energy provision under van Abel TH (1979) Submarine thermal springs on the anoxia remains to be investigated. Galapagos Rift. Science 203:1073-1083 Edmond JM, Measures C, McDuff RE, Measures C1 (1982) In conclusion, Riftia pachyptila is physiologically well Chemistry of hot springs on the East Pacific Rise and their adapted to survive the short-term oxygen fluctuations effluent di

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Edtonal responsibility: Otto Kinne (Editor), Submitted: February 17, 1998; Accepted: August 5, 1998 Oldendorf/Luhe, Germany Proofs received from authorjs): November 4. 1998