Mar Biol

DOI 10.1007/s00227-006-0338-9

RESEARCH ARTICLE

Effect of ambient oxygen concentration on activities of enzymatic antioxidant defences and aerobic metabolism in the hydrothermal vent worm, Paralvinella grasslei

Benjamin Marie · Bertrand Genard · Jean-François Rees · Franck Zal

Received: 14 December 2005 / Accepted: 19 April 2006© Springer-Verlag 2006

Abstract The alvinellid Paralvinella grasslei is a com-mon endemic polychaete from the deep-sea hydrother-mal vent communities located on the East PaciWc Rise(EPR). These organisms colonise a large range ofmicrohabitats around active sites where physico-chem-ical conditions are thought to generate reactive oxygenspecies (ROS). Furthermore, in this aerobic organism,ROS could also be generated by the activity of themitochondrial respiratory chain. In this paper, weinvestigated the eVect of ambient oxygen concentra-tion on the activities of three essential antioxidantenzymes (superoxide dismutase, SOD; catalase, CAT;glutathione peroxidase, GPX) and their relationshipswith the activity of enzymes involved in aerobic metab-olism (cytochrome c oxidase, COX; citrate synthase,CS). Results of incubation of P. grasslei in a high-pres-sure vessel with circulating seawater at diVerent oxy-gen partial pressures indicate that this worm regulatesCOX and CS activities diVerently in gills and bodywall. CAT and GPX activities increase in these tissues

when animals are maintained in Wltered surface seawa-ter. Moreover, levels of malondialdehyde increase ingills, testifying that oxidative damage occurs underthese conditions. CAT and GPX activities are posi-tively related to COX and CS activities, but no correla-tion was detected between SOD and the metabolicenzyme activities. In comparison with littoral annelids,SOD activities are very high whereas CAT activitiesare very low or absent in P. grasslei. The possible rea-sons for the occurrence of such diVerences are dis-cussed.

Introduction

Fauna inhabiting deep-sea hydrothermal vents arehighly endemic and have developed speciWc adapta-tions to cope with atypical and extreme physico-chemi-cal conditions (Childress and Fisher 1992). In additionto the elevated hydrostatic pressure and the completeabsence of sunlight shared by all deep-sea areas, thisenvironment is characterised by the resurgence of toxichydrothermal Xuids (Von Damm 1990). The speciesrestricted to deep-sea hydrothermal vents live in themixing zone between the hydrothermal Xuid and thedeep-sea water, and are distributed according to theirphysico-chemical tolerance to the Xuid (Desbruyèreset al. 1982; TunnicliVe 1991). Blum and Fridovich(1984) suggested that several toxic compounds of theXuid, in contact with the oxygen of deep-sea water,could produce reactive oxygen species (ROS) such assuperoxide anions (O2

•¡ ), hydrogen peroxide (H2O2)and hydroxyl radical (OH•), a highly toxic componentfor organisms. ROS can oxidise most cellular constitu-ents such as proteins, lipids or DNA and markedly

Communicated by S. A. Poulet, RoscoV

Jean-François REES and Franck ZAL are equally contributed to this work.

B. Marie · F. Zal (&)Equipe Ecophysiologie : Adaptation et Evolution Moléculaires, UPMC – CNRS UMR 7144, Station Biologique, BP 74, 29682 RoscoV cedex, Francee-mail: zal@sb-roscoV.fr

B. Genard · J. F. ReesInstitut des Sciences de la Vie,Université Catholique de Louvain, Animal Biology Unit, Croix du Sud 5, 1348 Louvain-la-Neuve, Belgium

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disturb vital cellular functions (Halliwell and Gutter-idge 1989).

To date, no data are known on the types andamounts of ROS around hydrothermal vents, but thereare many reasons to suspect the presence of ROS inthis ecosystem (Blum and Fridovich 1984; Dixon et al.2002). Several abiotic parameters are indeed favour-able to ROS formation. The occurrence of high con-centrations of metals, such as iron (II), reactingspontaneously with H2O2 produces highly toxic OH• byFenton’s reactions (Halliwell and Gutteridge 1986;Fridovich 1998; Stohs and Bagchi 1995). Radionuc-lides, such as radon that occur at high level in thehydrothermal habitats, could inXict oxidative damage(De Oliveira et al. 2001). Spontaneous oxidation of sul-phide in seawater produce ROS (Tapley et al. 1999)and sulphide was also been detected in organism’s tis-sues (Edmond et al. 1982; Cosson-Mannevy et al. 1988;Cherry et al. 1992; Cosson and Vivier 1997). High tem-peratures can as well favour the formation of O2

•¡

(Issels et al. 1986).As in other aerobic organisms, another potential

source of ROS is endogenously with the activity of themitochondrial respiratory chain which could continu-ously generate ROS at the rate of 1–2% of the totaloxygen consumed by animal tissues in resting condi-tions (Cadenas and Davies 2000), even if this phenome-non is today highly debated (Staniek and Nohl 2000;Keller et al. 2004). To cope with both endogenous andexogenous productions of ROS, organisms possess acomplex arsenal of antioxidative defence mechanismssuch as vitamins, glutathione, metallothioneins or spe-ciWc enzymes. The most important of these enzymes aresuperoxide dismutase (SOD) that converts O2

•¡ intoless toxic H2O2 via the reaction 2O2

•¡ + 2H + ! H2O2+ O2, catalase (CAT) which decomposes H2O2 intoH2O (2H2O2! 2H2O + O2), and glutathione peroxi-dase (GPX) which uses glutathione (GSH) as cofactorfor reducing peroxides according to 2GSH + ROOH!GSSG + ROH + H2O. In many cases, the levels ofantioxidant enzymes are known to be adjusted to thethreat posed by respiration-derived ROS. In mammals,increasing the maximal aerobic capacity by exerciseleads to increased levels of various antioxidant enzymes(Vincent et al. 2000). In deep marine Wshes SOD andGPX activities are apparently adjusted to maximumaerobic metabolism capacity (Janssens et al. 2000), asmeasured by cytochrome c oxidase (COX) and citratesynthase (CS) activities (Childress and Somero 1979;Thuesen and Childress 1993, 1994).

The alvinellid Paralvinella grasslei Desbruyères andLaubier 1982 is a common endemic polychaete fromhydrothermal vents of the East PaciWc Rise (Desbruyè-

res and Laubier 1991; Zal et al. 1995). Unlike other alv-inellids, P. grasslei does not secrete a tube but iscovered by an abundant mucus. Alvinellids are highlyaerobic organisms (Hand and Somero 1983; Hourdezet al. 2000), although adapted for life in hypoxiathrough optimised oxygen extraction in gills (Jouin andGaill 1990) and a high oxygen carryingcapacity (Toulmond et al. 1990). Indeed, this wormcolonises environments, like black smoker chimneyswhere it is exposed to low, highly Xuctuating, oxygenconcentrations (Johnson et al. 1988; Juniper and Marti-neu 1995). Intertidal polychaetes, which also live in aXuctuating oxygen environment, are well known toadapt their oxygen consumption and metabolismaccording to the oxygen availability (Toulmond andTchernigovtzeV 1984). Those worms down-regulatetheir ventilatory activity under hyperoxic conditions inorder to maintain their metabolic rates and to possiblylimit the toxic eVects of oxygen occurring during anover-oxygenation (Fox and Taylor 1955). Also, Xuctua-tions of oxygen levels are known to favour the forma-tion of ROS in animal’s tissues, a phenomenon leadingto the so-called ischemia–reperfusion injuries (e.g.Warner et al. 2004; Buja 2005). Therefore, one couldexpect that the low and unstable oxygen availability atvents could inXuence the aerobic metabolic rate as wellas the requirement for antioxidant enzymes.

For all the above reasons, one would Wrst expectP. grasslei to possess a well-developed and speciWcantioxidant defence system aimed to reduce the toxicROS occurring in the hydrothermal Xuid, and secondly,that its rate of oxidative metabolism enzymes and anti-oxidant system could be linked and have some ability torespond to variations in oxygen availability. To testthese hypotheses, we have analysed the activities ofantioxidant enzymes in P. grasslei and the long-termeVect of ambient oxygen concentration on antioxidantsand some oxidative metabolism enzymes, and lookedfor signs of oxidative damages in worms maintained inhyperbaric aquaria.

Materials and methods

Animal collection

Collection of P. grasslei was performed at Elsa (N 1248.1420–W 103 56.3060) and Parigo (N 12 48.5960–W103 56.4270) vent sites (13°N on the East PaciWcRise, t2,600 m of depth) during the PHARE cruise(May 2002), using the manipulated claw or the «slurpgun» of the ROV Victor 6000. Specimens werecollected on “Alvinella” habitat where usually the

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temperature is comprised between 10 and 30°C on thesurface of the colony. Animals were brought to the sur-face in watertight and temperature-insulated contain-ers. On board of the research ship N/O Atalante, theywere quickly transferred to a cold room (10°C) andanimals of around 5 cm of length were selected andcleaned of their mucus. For each in vivo experiments(cf. below), ten worms were dissected on ice and tissues(i.e. gills, gut and body wall) were frozen immediatelyin liquid nitrogen and stored at ¡80°C, to serve as ref-erence of physiological condition of the worm just afterthe decompression. Those “Freshly Caught” animalswere analysed and results compared to the worms ofthe reconditioning experiments. Seven other individu-als were immediately placed into perforated 50-ml Fal-con tubes for reconditioning experiments, which werestarted within 3 h of animal collection on hydrothermalsites.

In vivo exposure to diVerent partial pressures of oxygen

Animals just recovered on board, were immediatelyreconditioned in a high-pressure chamber pressurisedat 260 bars corresponding to habitat pressure at2,600 m depth. The reconditioning process was carriedout in the pressure vessel system Ipocamp, thermo-stated to 15°C (Shillito et al. 2001). Oxygen levels wereregulated upstream of Ipocamp using a chemical-parameter controller system (Syrene, described inChausson et al. 2004). Animals were placed inside thechamber containing Wltered seawater, re-pressurised(in 2 min), where the desired oxygen level was reachedafter 4–8 h of circulation at a Xow rate of approxi-mately 5 l h¡1, and then the worm was held at that levelfor 12 h. This experimentation time was chosen toallow physiological recovery of animals as recentlydemonstrated for the closely related species Alvinellapompejana (Shillito et al. 2004). The seawater pH wasmaintained between 6.2 and 6.5 (i.e. corresponding tothe pH found around the worms in situ) with a propor-tional pH controller (Consort R305, Inc.) connected totwo metering pumps injecting either 1 M NaOH or 1 MHCl. The experiments at in situ pressure were per-formed at three diVerent oxygen concentrations onthree set of diVerent worms (mean § SD): (1)“hypoxia” (12.8§6 �mol O2 l¡1 or 1§0.5 kPa or7.5§3.5 Torr); (2) “normoxia” (78§3 �mol O2 l¡1 or6.3§0.2 kPa or 50§2 Torr); (3) “hyperoxia” (203§9�mol O2 l¡1 or 16.2§0.8 kPa or 122§5 Torr). The ter-

minology used for the oxygen conditions was chosenaccording to oxygen concentrations found in the habi-tat of this worm (reviewed in Juniper and Martineu

1995, “hyperoxia” corresponding to air-saturated sea-water). Oxygen concentrations were measured at theoutlet of the pressurised chamber with an oxymeter(WTW oxi) throughout the experiments.

Sample preparation

Tissues (i.e. gill, body wall and gut) were dissected onice, frozen in liquid nitrogen and kept at ¡80°C untilanalysed (i.e. around 6 months after collection). Tis-sues were weighed and homogenised (w/v=1:5) inphosphate buVered saline (PBS), pH 7.4, containing1% Triton X-100. An aliquot of the homogenate wasused for the determination of the protein content(Lowry et al. 1951), and the remaining was centrifugedat 15,000g (15 min, 4°C). The supernatant was used forenzyme activity assays as well as for the analysis of themalondialdehyde (MDA) contents. Enzymatic activitymeasurements were performed at 25°C in order toallow comparisons with previous investigations, andbecause hydrothermal species are predicted to havemaximal sustained body temperature between 20 and30°C (DahlhoV and Somero 1991).

Catalase assay (EC 1.11.1.6)

In a Wrst tentative experiment, we attempted measur-ing catalase activity (CAT) using the classical spectro-photometrical method reported by Blum and Fridovich(1984), but no activity was detected by this assay. Con-sequently, CAT was measured by chemiluminescenceaccording to the method of Maral et al. (1977) modi-Wed for 96-well microplates by Janssens et al. (2000).The consumption of H2O2 by CAT was followed at25°C on a PC-controlled microplate luminometer(Berthold LB96 P). Fifty microlitre of 1 �mol l¡1 H2O2was added to 50 �l of sample diluted in 100 �l of phos-phate buVer 100 mmol l¡1, pH=7.8, containing0.6 mmol l¡1 EDTA. After a 30-min incubation at25°C, the injection of 50 �l of 20 mmol l¡1 luminol and11.6 U ml¡1 horseradish peroxidase produces an emis-sion of light. The light intensity was assumed to be pro-portional to the remaining quantity of H2O2. CATactivity in samples was quantiWed by constructing stan-dard curves using puriWed bovine liver CAT dissolvedin PBS-Triton buVer and expressed in internationalunits (�mol H2O2 consumed min¡1).

Superoxide dismutase assay (EC 1.15.1)

The spectrophotometric method of Flohé and Ötting(1985) was adapted for microplate measurements. Theassay is based on the competition between superoxide

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dismutase (SOD) and oxidised cytochrome c for O2•¡

generated by the reaction of hypoxanthine with xan-thine oxidase (EC 1.1.3.22). Reduction rate of cyto-chrome c (2 �mol l¡1) was measured at 550 nm (25°C),in 180 �l of phosphate buVer (50 mmol l¡1, pH=7.8)with 0.5 mmol l¡1 EDTA, 5 �mol l¡1 hypoxanthineand 10 �l diluted sample. The reaction was initiated byinjecting 10 �l xanthine oxidase (0.2 U ml¡1). SODactivity in samples was estimated with Cu, Zn-SODpuriWed from bovine erythrocytes and expressed ininternational units (1 U corresponds to the SODamount inhibiting by 50% the rate of cytochrome creduction).

Glutathione peroxidase assay (EC 1.11.1.9)

Glutathione peroxidase was measured spectrophoto-metrically as reduction rate of NADPH at 340 nm(25°C), using the protocol of Paglia and Valentine(1967) modiWed for 96-well microplates. Fifteen micro-litre of diluted sample was added to 120 �l Tris–HClbuVer (50 mmol l¡1, pH=7.6) containing 0.1 mmol l¡1

EDTA, 0.14 mmol l¡1 NADPH, 1 mmol l¡1 glutathi-one and 1 U glutathione reductase (EC 1.6.4.2). Thereaction was initiated by the addition of 15 �l t-butylhydroperoxide 0.2 mmol l¡1. GPX activity was esti-mated using the molar extinction coeYcient ofNADPH (6.220 mol l¡1 cm¡1) and expressed in inter-national units (�mol NADPH consumed min¡1).

Aerobic metabolism estimates

Aerobic metabolic capacity was estimated from citratesynthase (EC 4.1.3.7) and cytochrome c oxidase activi-ties (EC 1.9.3.1), two mitochondrial enzymes well cor-related to the oxygen consumption in vertebrates andinvertebrate tissues (Childress and Somero 1979;Thuesen and Childress 1993, 1994).

Citrate synthase activity was measured according tothe protocol of Sheperd and Garland (1969) modiWedfor multiwell plates. Twenty microlitre of diluted sam-ple were added to 160 �l of Tris–HCl buVer (100 mmoll¡1, pH=8.0) containing 15 �mol l¡1 of 5,5�-dithio-

bis(2-nitrobenzoic acid) (DTNB) and 25 �mol l¡1 ace-tyl-CoA. Consumption of acetyl-CoA was followed at420 nm after adding 20 �l oxaloacetate 50 �mol l¡1

(25°C). CS activity was estimated using the molarextinction coeYcient of DTNB (13.600 mol l¡1 cm¡1)and expressed in international units (�mol DTNBconsumed min¡1).

The spectrophometric assay method for cytochrome coxidase (COX) of Hand and Somero (1983) was adaptedfor microplates. The oxidation of 0.1 mol l¡1 cytochrome

c solubilized in 180 �l of Tris–HCl buVer (50 mmol l¡1,pH=7.6) was followed at 550 nm (25°C) after addition of20 �l of diluted sample. COX activity was estimatedusing the molar extinction coeYcient of cytochrome c(19.600 mol l¡1 cm¡1) and expressed in internationalunits (�mol cytochrome c consumed min¡1).

Determination of malondialdehyde (MDA)

The occurrence of oxidative damage in tissues wasinvestigated by assaying MDA that forms upon ROSattacks on unsaturated lipids (Storey 1996). The con-centration of MDA was estimated in gills and bodywall of P. grasslei according to Yagi’s method (1984)adapted for microplates. Thirty microlitre of samplewere added to 48 �l of thiobarbituric acid (0.67%) in0.3 mol l¡1 NaOH and 24 �l of trichloroacetic acid at15%. After 30-min incubation of microplates with aTeXon lid at 95°C and cooling to room temperature,100 �l butanol was added. Microplates were centri-fuged for 5 min (800g, 4°C) and the Xuorescence wasmeasured at 555 nm (excitation 515 nm) with a Xuo-rimeter Ascent (Labsystems). MDA concentration wasestimated from a standard curve made with commer-cial MDA.

Reagents

All reagents were purchased from Sigma (St Louis,USA), except for Triton X¡100, MDA, GSH and glu-tathione reductase obtained from Boehringer (Mann-heim, Germany).

Statistical analyses

The enzymatic activities and the concentrations weremeasured in triplicate and are presented as means of asample size of 7. Means are related to protein contentand wet mass of the tissue. Although the expression ofenzyme activities as a function of the wet mass seemsto be more appropriate for comparing physiologicalcapacities of the tissues, activities expressed relativelyto the protein content are often used in works pub-lished on diVerent species.

Mean comparisons were carried out using Statview 4.5software. We used a non-parametric Kruskal-WallisU-test and P<0.05 was accepted to indicate a signiWcantdiVerence. To evaluate correlation between enzymeactivities in each tissue, linear regressions were carriedout on all experimental groups taken separately. Correla-tion tests were also carried out for each tissue on all datacombined in order to consider co-variation of enzymeactivities during experiments. Untransformed data were

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used to describe the relation between COX and CS activ-ities or on log-transformed data for antioxidant and met-abolic enzyme relationship, see Janssens et al. (2000).Furthermore, simple linear regression tests were carriedout using SAS software, considering the group eVect asan external variable [i.e. “Freshly Caught” (FC),hypoxia, normoxia, hyperoxia]. Correlation was declaredsigniWcant when P value was less to 5%.

Results

Antioxidant and mitochondrial enzymes in P. grasslei

Activities of all enzymes (U g¡1 wet weight) in tissuesfrom “Freshly Caught” animals (named “FC” in thefollowing text) are shown in Table 1. CAT, SOD,GPX, COX and CS activities were detected in gills, gutand body wall of P. grasslei.

The highest antioxidant activities for all enzymeswere measured in the gut and in the body wall,although some of these activities are not signiWcantlydiVerent in comparison with the gills (Table 1). Thegills show the highest CS level, almost twice the valuefound for the gut or the body wall. COX activity wassimilar in the gut and the gills, and was lower in bodywall. Surprisingly, CS/COX ratios were high: approxi-mately 5 in gills, 3 in the body wall and 2 in the gut, forcomparison purpose this ratio is comprise between 0.1and 0.4 in Wsh tissues (Lucassen et al. 2003).

EVects of conditioning at various oxygen concentrations on aerobic metabolism activities

Worms brought to the surface and reconditioned inpressure chambers were quite active and exhibitedparapodia movements after the experiments. The con-ditioning of P. grasslei at diVerent oxygen concentra-tions had important eVects on COX and CS activities in

the gills and the body wall, but not in the gut (Table 2).However, responses were diVerent in gills and bodywall tissues. Figure 1 shows the eVect of oxygen con-centration on COX and CS activities (U g¡1 wetweight) in gills. Both COX (Fig. 1a) and CS (Fig. 1b)activities were lower of FC values (P<0.01) after condi-tioning in hypoxia (12§6 �mol O2 l¡1). In normoxia(78§3 �mol O2 l¡1), COX and CS activities increased(P<0.01) in comparison with hypoxia values. In hyper-oxia (203§9 �mol O2 l¡1) conditions, COX and CSactivities decreased (P<0.001 and P<0.05, respectively)below FC values. Figure 2 reports the inXuence of oxy-gen concentration on COX and CS activities in thebody wall (U g¡1 wet weight). While conditioningunder hypoxia and normoxia had no signiWcant eVecton COX activity in comparison with FC, COX activityslightly increased in hyperoxia conditions when com-pared to normoxia (P<0.05). Similarly, CS activity alsoincreased under hyperoxia conditions.

Figure 3 shows the linear correlation between COXand CS activities (U g¡1 wet weight) in gills and bodywall tissues. When the diVerent groups were consid-ered separately, no signiWcant correlation betweenboth enzymes was observed in gills. However, a highlysigniWcant relationship between CS and COX wasfound when data of all experiments were pooledtogether, emphasising that COX and CS activities ingills are similarly aVected by exposure to diVerent oxy-gen concentrations. In body wall, signiWcant correla-tions were observed in FC, hypoxia and hyperoxia withquite similar slope coeYcients; the relationshipobserved in bulk data also indicates a correlationbetween COX and CS activities in this tissue. Theseresults show that, despite of the diVerent responses tooxygen concentration in gills and body wall tissues,COX and CS activities were linearly and positively cor-related in both tissues (R=0.67, P<0.001 and R=0.43,P<0.05, respectively, for all clump data). The slopescharacterising their relationship were similar in both

Table 1 Antioxidant and aerobic metabolism activities in diVerent tissues of Paralvinella grasslei « Freshly Caught » (U g–1 wet weight)

All enzymatic activities were measured at 25°C

SigniWcantly diVerent from the activity measured in gut (*P<0.05, **P<0.0001)

SigniWcantly diVerent from the activity measured in body wall (***P<0.05, ****P<0.001)

Paralvinella grasslei Antioxidative enzymes Aerobic metabolism enzymes

CAT SOD GPX COX CS

Units g¡1 wet weight [Mean § SE (n=7)]

Gills 5.9§0.2** 920§185 0.54§0.03* *** 1.08§0.32 5.4§0.8** ****Gut 21.2§2.8**** 1377§644 0.78§0.11 0.98§0.22 1.9§0.4Body wall 9.0§2.4 1367§659 0.81§0.20 0.76§0.18 2.3§0.2

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tissues (CS=1.92 COX + 2.99, in gills; CS=2.00COX + 1.50, in body wall).

Antioxidant activities related to the oxygen concentration

Figure 4 shows the eVects of diVerent oxygen concen-trations on CAT and GPX activities in gills and bodywall during three acclimation experiments. In gills, adecrease of CAT activity was observed (P<0.01) afterhypoxic conditioning (Fig. 4a) in comparison to FCgroup. CAT activity was signiWcantly higher in norm-oxia when compared to hypoxia (P<0.01). DiVerently,GPX activity in body wall tissues was aVected by theoxygen concentration (Fig. 4b) as it increased morethan twice after 12-h exposure to high oxygen(P<0.001), while no eVect was observed at loweroxygen concentration. In another way, SOD activity

seems not to be aVected by the diVerent oxygen treat-ment and does not show any signiWcant diVerence inboth tissues (data not shown).

Relationships between antioxidant and aerobic metabolism activities

Figure 5a, b shows the relationships between CAT andCOX activities in gills and between GPX and CS in bodywall tissues (expressed on Log/Log axes), respectively.No signiWcant correlation was observed within the diVer-ent groups taken separately. However, signiWcant rela-tionships were found within experiments (bulk data,considering the eVect of the group as an external factor),underlining the link between CAT and COX activities ingills, and GPX and CS activities in body wall. Theserelationships are best described as exponential increasesin antioxidant enzymes with increasing CS or COXactivity (U g¡1 wet weight) following the equationsCAT=0.16 COX0.98 (P<0.001) and GPX=2.84 CS0.27

(P<0.01) (Fig. 5b). Contrarily to what we observed for

Fig. 1 Paralvinella grasslei. Aerobic metabolism activities (U g¡1

wet weight) in gills after incubations at various ambient oxygenconcentrations. Animals were incubated at 260 bar, 12 h in Wl-tered sea-water maintained at 15°C once the oxygen conditionswere stabilised, in hypoxia (12§6 �mol O2 l¡1), normoxia(78§3 �mol O2 l¡1) and hyperoxia (203§9 �mol O2 l¡1) (seematerials and methods). a COX activity in gills. b CS activity ingills. * SigniWcantly diVerent from the value at the inferior oxygenconcentration point (*P<0.05, **P<0.01, ***P<0.001). Values aremeans § SD (n=7); + signiWcantly diVerent from FC value(+P<0.05, ++P<0.01, +++P<0.001). Kruskal-Wallis, non-parametricU-test

0

0.5

1

1.5

2

FC Hypoxia Normoxia Hyperoxia

CO

X a

ctiv

ity (

units

g-1 w

et w

eigh

t)

A

++

+ ✻✻

+++ ✻✻

0

2

4

6

8

FC Hypoxia Normoxia Hyperoxia

B

++

✻✻

+ ✻

CS

activ

ity (

units

g-1 w

et w

eigh

t)

Fig. 2 Paralvinella grasslei. Aerobic metabolism activities (U g¡1

wet weight) in body wall after incubations at various ambient oxy-gen concentrations like previously described. a COX activity inbody wall. b CS activity in body wall. * SigniWcantly diVerent fromthe value at the inferior oxygen concentration point (*P<0.05).Values are means § SD (n=7); + signiWcantly diVerent from FCvalue (+P<0.05, ++P<0.01, +++P<0.001). Kruskal-Wallis, non-para-metric U-test

0

1

2

3

4

FC Hypoxia Normoxia Hyperoxia

CS

acti

vity

(un

its

g-1 w

et w

eigh

t) B++

+

++ ✻

0

0.5

1

FC Hypoxia Normoxia Hyperoxia

A

CO

X a

ctiv

ity

(uni

ts g

-1 w

et w

eigh

t)

✻

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CAT and GPX, no relationship between SOD and COXneither between SOD and CS (data not shown) could befound in any tissue whatever data were expressed rela-tively to the protein content or the wet weight of the tis-sue, as revealed for the gills (Fig. 5c).

Induction of lipid peroxidation

Figure 6 shows eVects of ambient oxygen level in theaquarium on MDA levels in gills and body wall tissues

of P. grasslei. In FC specimens, MDA levels werehigher in gills (6.5§0.8 nmol g¡1 wet weight) than inbody wall (3.8§0.3 nmol g¡1 wet weight). The incuba-tion of animals in hyperoxia increased (P<0.01) thelevel of MDA in gills (12.6§0.9 nmol g¡1 wet weight),about two folds that measured in FC animals, testifyingthat some oxidative damage may have occurred in thistissue. Quite remarkably, no variation of MDA con-centrations were observed in body wall tissues afterany reconditioning experiments.

Discussion

Since free radicals are known to be produced continu-ously in all aerobic cells during the electron transfer pro-cess in mitochondria, the positive correlation betweenGPX and CS in body wall tissues (Fig. 5b) suggests thatlevels of GPX are adjusted to the peroxides generateddirectly or indirectly by oxidative phosphorylation. Suchcorrelations have been demonstrated in other organ-isms, such as Wsh (e.g. Janssens et al. 2000; Morales et al.2004) and mammals (Leeuwenburgh et al. 1997). Fur-thermore, it seems that COX and CS are co-regulated astheir levels vary in parallel to each other in both gills andbody wall tissues (Figs. 1, 2, 3). This co-variation of thoseenzyme activities could suggest a regulation of mito-chondrial density, modifying the oxidative capacity ofthe tissues, as suggested for Arenicola marina by Som-mer and Pörtner (2002).

Blum and Fridovich (1984) reported the presence ofSOD and GPX activities in the tube-worm Riftia

Fig. 3 Paralvinella grasslei. Linear relationships between COX(U g¡1 wet weight) and CS (U g¡1 wet weight) in gills and bodywall. In gills, no signiWcant correlation was observed within thediVerent groups taken separately. The dotted line Wts the equationof the co-variation tendency of within experimentations (bulk da-ta): CS activity=1.92 COX activity + 2.99, R=0.67, P<0.001(n=26). In body wall, signiWcant correlations were observed inFC, hypoxia and hyperoxia (data not shown). Bulk data alsoshows similar relationship than in gills (dotted line): CSactivity=2.00 COX activity + 1.50, R=0.43, P<0.05 (n=27)

0

2

4

6

8

10

0 0.5 1 1.5 2

GillsBody wall

COX activity (units g-1 wet weight)

CS

acti

vity

(un

its

g-1 w

et w

eigh

t)

Table 2 Antioxidant and aerobic metabolism activities of three tissues coming from the hydrothermal vent species Paralvinella grassleisubmitted at three oxygen partial pressures (U g¡1 of wet weight)

All enzymatic activities were measured at 25°C and values are the means and standard deviations of 7 diVerent samples

Hypoxia=12.8§6 �mol O2 l¡1 or 1§0.5 kPa or 7.5§3.5 Torr

Normoxia=78§3 �mol O2 l¡1 or 6.3§0.2 kPa or 50§2 Torr

Hyperoxia=203§9 �mol O2 l¡1 or 16.2§0.8 kPa or 122§5 Torr

Paralvinella grasslei CAT SOD GPX COX CS

Units g¡1 wet weight [Mean § SE (n=7)]

HypoxiaGills 1.28§0.46 639.33§371.55 0.64§0.11 0.17§0.11 2.65§0.30Gut 12.11§7.45 1597.83§454.44 0.52§0.20 0.31§0.12 0.67§0.50Body wall 7.98§2.35 2307.50§566.50 0.98§0.13 0.66§0.10 3.07§0.28

NormoxiaGills 4.31§1.30 1287.66§464.00 0.66§0.16 1.56§0.14 6.12§1.32Gut 17.13§3.88 1999.50§1209.50 0,63§0.13 0.87§0.29 1.76§0.61Body wall 8.15§0.48 2245.50§538. 60 0.85§0.12 0.66§0.06 2.94§0.28

HyperoxiaGills 4.08§1.65 1840.28§743.75 0.84§0.27 0.33§0.10 3.78§0.83Gut 9.53§3.42 2142.33§698.88 0.47§0.01 0.15§0.06 0.37§0.20Body wall 8.26§3.88 2815.75§648.43 2.26§0.20 0.80§0.09 3.63§0.47

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pachyptila and the giant clam Calyptogena magniWca,but could not detect any CAT activity in either species.Generally, CAT is a major antioxidant and in combina-tion with GPX detoxiWes H2O2, formed by SOD andother exogenous sources. However, using a more sensi-tive luminometric method operating at low H2O2 con-centration (Maral et al. 1977 modiWed by Janssenset al. 2000), we were then able to detect very low levelsCAT-like activity in all tissues. CAT-like activity inbody wall of P. grasslei is very low (0.21§0.13 U mg¡1

protein) in comparison to Arenicola marina(8§2 U mg¡1 of protein) which possesses the lowestCAT activity amongst non-hydrothermal vent annelids(Table 3). Such low catalase levels suggest that thisenzyme could be absent in P. grasslei tissues and thatthe occurrence of this “catalase-like” activity may reX-ect the occurrence of H2O2-scavengers such as antioxi-dant osmolytes or other enzymes. Interestingly, Yinet al. (2000) found high concentration of potential anti-oxidant osmolytes like hypotaurine in hydrothermalvent species. As another explanation, the consumptionof H2O2 could be related to Fenton reactions catalysedby metals likely to be present in tissue homogenates. In

contrast, a high SOD activity was measured: to date,P. grasslei possesses the highest SOD activity(30§15 U mg¡1 of protein in body wall) in comparisonto hydrothermal vent (R. pachyptila 9.3 U mg¡1 of

Fig. 4 Paralvinella grasslei. Antioxidant activities (U g¡1 wetweight) in gills (a) and body wall (b) after incubations at variousambient oxygen concentrations. a CAT activity. b GPX activity.Values are means § SD (n=7); * signiWcantly diVerent from thevalue at the inferior oxygen concentration point (**P<0.01);+ signiWcantly diVerent from FC value (+P<0.05, ++P<0.01,+++P<0.001). Kruskal-Wallis, non-parametric U-test

0

2

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6

FC Hypoxia Normoxia HyperoxiaCA

T a

ctiv

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et w

eigh

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ctiv

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(uni

ts g

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et w

eigh

t)

B+++ ✻✻

Fig. 5 Paralvinella grasslei. Relationships between antioxidantactivities (U g¡1 wet weight) and aerobic metabolism activities(U g¡1 wet weight) in gills and body wall. a Exponential relation-ship between CAT and COX in gills. No signiWcant correlationwas observed within the diVerent groups taken separately. Thedotted line Wts the equation of the co-variation tendency withinexperimentations (bulk data): COX activity=¡0.80(CAT activ-ity)0.98, R=0.65, P<0.001 (n=26). b Exponential relationship be-tween GPX and CS in body wall. No signiWcant correlation intodiVerent group take separately was observed. The dotted line Wtsthe equation of the co-variation tendency of within experimenta-tions (bulk data): CS activity=0.44(GPX activity)0.29, R=0.60,P<0.01 (n=27). c No signiWcant relationship was detected be-tween SOD activity and aerobic metabolism activities, e.g. COXactivity both in gills and in body wall

0.01

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protein in muscle) or non-hydrothermal vent annelids(Heteromastus Wliformis 17§6 U mg¡1 of protein in bodywall). Thus, P. grasslei possesses a low CAT-like activityand a high SOD activity in comparison to other annelids.Since Dixon et al. (2002) have shown that the sensitivityof P. grasslei to exogenous H2O2 was similar to thatrecorded for shallow-water polychaetes and that GPXlevels are similar to those found in other annelids, it ispossible that the detoxiWcation of SOD-derived H2O2 canbe taken over other peroxidases. Another interestingWnding is that H2O2 could also be detoxiWed by chemicalreaction with H2S (Millero et al. 1989). Consequently, itwould be judicious in future studies to measure H2O2directly in the environment and inside the organisms andto look for other peroxidases in P. grasslei.

Quite surprisingly, no correlation between SODactivity and oxidative metabolism enzymes wasdetected. This absence of relationship seems to indi-cate that the high SOD activity could be mainly aimedat superoxide anions generated through respiration-independent processes. The high metal content in tis-sues of P. grasslei (e.g. Cd, Fe, Hg and As; Cosson andVivier 1997; Desbruyères et al. 1998), could be animportant source of superoxide anions. Highest SOD

activities were associated with gut tissues in compari-son to body wall and gills tissues (Table 3). This gut tis-sue comprises the digestive tract with its content, and

Table 3 Antioxidant and aerobic metabolism activities of hydrothermal and non-hydrothermal annelids (U mg–1 of protein)

All enzymatic activities were measured at 25°C and all protein concentrations were determined from homogenized tissue, except for (a)which activities were measured at 20°C and protein concentrations assayed from supernatant extracts

ND Not detectable, numbers in parentheses are the samples size

Species Antioxidative enzymes Aerobic metabolism enzymes Source

CAT SOD GPX COX CS

Units mg¡1 of protein [Mean § SE (number of specimens)]

Hydrothermal vent annelids

Paralvinella grasslei

Gills 0.16§0.01 (7) 24§5 (7) 0.014§0.001 (7) 0.028§0.009 (7) 0.142§0.021 (7) This studyGut 0.82§0.11 (7) 53§25 (7) 0.030§0.004 (7) 0.038§0.008 (7) 0.073§0.015 (7)Body wall 0.20§0.05 (7) 30§15 (7) 0.018§0.004 (7) 0.017§0.004 (7) 0.051§0.004 (7)

Riftia pachyptila Muscles n.d. (1) 3.7 (1) 0.006 (1) – – Blum and Fridovich(1984)

Alvinellapompejana

Gills – – – 0.08 (1)a – Hand and Somero (1983)Body wall – – – 0.05 (1)a –

Non-hydrothermal vent annelids

Arenicola marina Body wall 8§2 (5) 6§3 (5) – – – Buchner et al. (1996)

Nereis diversicolor Body wall 57§14 (10) 3§1 (10) – – – Abele-Oeschger et al. (1994)

Laeonereis acuta Body wall 2.2§0.4 (4) 3.5§0.5 (4) – – – Geracitano et al. (2004)

Heteromastus Wliformis

Body wall 21§6 (7) 17§6 (6) – – – Abele et al. (1998)

Phyllodoce mucosa Body wall – 7§1 (8) – – – Abele-Oeschger (1996)

Eisenia foetida Body wall 91§4 (3) – 0.013§0.004 (3) – – Saint-Denis et al. (1998)

Fig. 6 Paralvinella grasslei. Concentration of malondialdehyde(nmol g¡1 wet weight) in gills and in body wall after incubations atvarious ambient oxygen concentrations. Animals were incubated at260 bar, 12 h in Wltered sea-water maintained at 15°C once the oxy-gen conditions were stabilised, in hypoxia (12§6 �mol O2 l¡1),normoxia (78§3 �mol O2 l¡1) or hyperoxia (203§9 �mol O2 l¡1).Values are means§SD (n=3 individuals). * SigniWcantly diVerentfrom the value at the inferior oxygen concentration point; + signiWcantly diVerent from FC value (**P<0.01) t-test

0

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the chloragog tissue. Buchner et al. (1996) have foundSOD activity fourfold higher in the chloragog thanin body wall tissues of the intertidal polychaete A. marina.These authors have proposed that chloragog wasconfronted to a large quantity of free radicals resultingfrom hemoglobin autoxidation. In addition, comparingSOD levels in marine invertebrates, Abele-Oeschger(1996), have observed that high SOD activities areoften linked to the presence of hemoglobin and havesuggested that these respiratory pigments have atendency to form superoxide anions via autoxidation(Misra and Fridovich 1972; Abele-Oeschger andOeschger 1995; Hermes-Lima et al. 1988). It is impor-tant to note that P. grasslei possesses a large amount ofintra and extracellular hemoglobin (Toulmond et al.1990; Zal et al. 2000). Another interesting hypothesissuggested by Abele-Oeschger (1996) is that SOD activ-ity in benthic invertebrates could be related to in situH2S concentrations like those found in the environ-ment of P. grasslei.

Malondialdehyde concentration in P. grasslei was inrange of those observed in other invertebrates (Her-mes-Lima et al. 1988), suggesting that no extensive oxi-dative damage to lipids occur in FC specimens.However, MDA levels in gills seem to indicate that theantioxidant arsenal could be insuYcient in this issuebut not in body wall high oxygen conditions. Indeed,accumulation of MDA occurred in gills but not in bodywall tissues when P. grasslei is exposed to hyperoxicconditions (Fig. 6). The higher susceptibility of gillsmight have to do with the fact that these are directlyexposed to ambient oxygen and act as a diVusion bar-rier for oxygen into other tissues, which could beexposed to lower O2 levels. Another consequence ofthis result is that it demonstrates the occurrence of oxi-dative stress in P. grasslei submitted to air-saturatedsurface seawater. This observation is not highly sur-prising since ecological data indicate that deep-seahydrothermal vent organisms living on chimney emit-ting anoxic Xuids usually breath in poorly oxygenated(5 �mol l¡1) seawater (Di Meo-Savoie et al. 2004), andexperience maximum oxygen concentrations of120 �mol l¡1 (Johnson et al. 1988). This seems to con-Wrm work by Dixon et al. (2002) who observed highlevels of DNA damage in P. grasslei conditioned 12 hat 260 bar in air-saturated surface seawater. Thus thisfurther indicates that low oxygen concentration is nec-essary for keeping these animals in physiological condi-tions.

Similarly, the intertidal polychaete HeteromastusWliformis, which was shown to never encounter morethan 150 �mol O2 l¡1 in its natural biotope, is known towaste away under well-aerated conditions (Abele et al.

1998). As a possible evidence that surface oxygen con-centrations could be toxic to hydrothermal organisms,the alvinellid Alvinella pompejana that colonises aneven more anoxic part of the hydrothermal ecosystemthan P. grasslei (Jollivet et al. 1995; Juniper and Marti-neu, 1995) had never been kept alive even for a shortperiod in hyperbaric aquaria circulated with air-satu-rated seawater. Only recently, some A. pompejanawere maintained in a pressure vessel (Ipocamp) aliveand this coincided with the reduction of oxygen pres-sure down to levels comparable to the hypoxic condi-tions of the present study (Shillito et al. 2004). It is thuspossible that the speciWc adaptations allowing this ani-mal to maintain normal aerobic metabolism at very lowoxygen concentration (i.e. low P50-hemoglobin, highbranchial surface and presence of an internal oxygenreservoir, Hourdez et al. 2000) could lead to a highersusceptibility to oxygen toxicity.

In conclusion, our results indicate that P. grassleican regulate its enzymatic aerobic metabolism capacityand antioxidant enzymes according to the ambient oxy-gen concentration. During our experiments, when theoxygen concentration exceeded levels normally persist-ing in the environment of the worms, GPX activityincreased in body wall concomitantly to an increase ofaerobic enzyme activities, while CAT-like activity didnot change compared to in situ, on the other hand, aer-obic enzyme activities declined in the gills. These mod-iWcations were accompanied by signs of lipidperoxidation in gills, indicating that the upregulationsof GPX and CAT-like activities could not preventoxidative stress to occur. The high SOD activity inP. grasslei tissues and the absence of correlationbetween the level of this enzyme and the aerobicmetabolism indicators suggest that this enzyme couldtarget high superoxide anions levels related to non-respiratory mechanisms. On the other hand, the verylow CAT-like and the average GPX activities are sug-gestive of the presence of other mechanisms dealingwith H2O2 detoxiWcation.

Acknowledgements The authors would like to thank the cap-tain and the crews of the N/O L’Atalante and ROV Victor 6000.We are also very grateful to Nadine Le Bris and Françoise Gaill,the chief scientists of the French research cruise PHARE’02. Wethank Dominique Davoult for his advice on statistical treatments.We would also like to thank Bruce Shillito for the experimentsrealized in IPOCAMP and Cécile Marchand for assistance in thelaboratory. We thank the EAEM team from RoscoV for their ad-vises and comments on this work. We are also very grateful to thefour anonymous referees for their useful comments and remarks,which considerably improve the submitted form of this manu-script. These studies were supported by the French Ministère desAVaires Etrangères under the Integrated Program Action calledTournesol (N° 05365 GT) and the Fonds de la Recherche Fonda-mentale Collective (FRFC) convention 2.4595.06.

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