Temperature resistance of Hesiolyra bergi, a polychaetous annelid living on deep-sea vent smoker walls

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Temperature resistance of Hesiolyra bergi, a polychaetous annelid living on deep-sea vent smoker walls
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  MARINE ECOLOGY PROGRESS SERIESMar Ecol Prog SerVol. 216: 141–149, 2001Published July 6 INTRODUCTION Over the last decade, spectacularly high temperaturemeasurements at the East Pacific Rise (EPR) hydro-thermal vents have suggested that some invertebratesliving on vent chimney walls could experience habitattemperatures far beyond the upper limits usual for sus-tained survival of most biota. One specimen of the poly-chaetous annelid Alvinella pompejana was observedcurling around a temperature probe indicating 105°C,while 6 other discrete recordings inside these annelids’tubes indicated values in the 40 to 80°C range (Cheval-donné et al. 1992). These observations showed thatvent creatures could survive exposure, atleast briefly,to very high temperatures. However, a more recentstudy based on 6 independent time-series temperaturemeasurements, again inside the tubes of these worms,increased the thermal tolerance limits of this biota:these recordings suggested that A. pompejana maysurvive temperatures of up to 60°C for several hours(Cary et al. 1998). In comparison, the ostracod Potamo-cypris  sp., which occurs in hot springs of North Amer-ica, showed 100% survival after 5 h at 49°C, but only33% after 12 h (Wickstrom & Castenholz 1973). There-fore, for this organism, considered one of the most ther-motolerant aquatic organisms by Chevaldonné et al.(2000), prolonged survival is not possible above 49°C.Such data inevitably pose the question of temperaturetolerance of these ‘hot pole’ hydrothermal vent species(Fisher 1998, Chevaldonné et al. 2000), especially sincebiochemical data (Somero 1992) suggest that thesesame thermophilic species may not survive prolongedexposure to temperatures above 40 to 50°C (Dahlhoff &Somero 1991, Dahlhoff et al. 1991, Childress & Fisher © Inter-Research 2001*E-mail: bruce.shillito@snv.jussieu.fr Temperature resistance of Hesiolyra bergi  , apolychaetous annelidliving on deep-sea ventsmoker walls Bruce Shillito 1, *, Didier Jollivet 2 , Pierre-Marie Sarradin 3 , Philippe Rodier 3 , François Lallier 2 , Daniel Desbruyères 3 , Françoise Gaill 1 1 Unité Mixte de Recherche 7622 (UMR), Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie Moleculaire et Cellulaire du Developpement (LBMCD), Marine Biology Group, University of Paris (UPMC), 7 Quai St Bernard, Batiment A, 75252 Paris Cedex 5, France 2 Roscoff Marine Station, Unité Propre de Recherche (UPR) 9042, Centre National de la Recherche Scientifique (CNRS), Ecophysiology Group, BP 74, 29682 Roscoff Cedex, France 3 Institut Français de Recherche Pour l’Exploitation de la Mer (IFREMER) Centre de Brest, DRO-EP, BP 70, 29280 Plouzané, France ABSTRACT: For the first time, in vivo  heat-exposure experiments were conducted on the hydro-thermal vent polychaete Hesiolyra bergi  from the hottest part of the vent biotope. Using a pressurisedincubator equipped with video-facilities, we found that H. bergi  , which forages around and in thetubes of the thermophilic Alvinella sp., became hyperactive once temperature exceeded 35°C andfurther lost co-ordination in the 41 to 46°C interval, just before death occurred. Another exposureexperiment at 39°C for 3 to 4 h led to 80% mortality (max) 9 h after heat shock, and 100% thereafter.In view of the much higher temperatures recorded in this organism’s habitat, these results suggestthat tolerance to high temperatures (exceeding 40°C) is not a pre-requisite for life amongst alvinellidtubes. Behavioural responses (escape from heat) may suffice.KEY WORDS: Hydrothermal vents · Behaviour · Adaptation to heat · IPOCAMP Resale or republication not permitted without written consent of the publisher   Mar Ecol Prog Ser 216: 141–149, 2001 1992, Gaill et al. 1995, Jollivet et al. 1995, Juniper &Martineu 1995, Desbruyères et al. 1998). Clarificationwould require in situ recordings of the body tem-perature of these hot-environment species, a difficultprospect at deep-sea vents (Desbruyères et al. 1998,Fisher 1998). An alternative approach is heat-resis-tance experiments with live animals in simulated envi-ronmental conditions. Provided that animals survivethe collection trauma and that appropriate means ofevaluating their condition (physiological, behavioural)are available, the question of the temperature resis-tance of vent animals may be directly addressed bysubmitting them to various temperature regimes.Here we present data based on video-observationsof vent worms incubated at in situ pressure, usinganewly designed pressurized incubator:IPOCAMP(Incubateur Pressurisé pour l’Observation et la Cultured’Organismes Marins Profonds; see Fig. 1a). For thefirst time, heat-resistance investigations have beencarried out on live invertebrates endemic to the hottestpart of the hydrothermal vent biotope: the wall ofactive vent chimneys. Hesiolyra bergi  , the ‘caterpillarworm’ (Blake 1985, Desbruyères et al. 1985, 1998), isan EPR-vent polychaete annelid (Desbruyères & Se-gonzac 1997), that attains a maximum length of 6 to7cm (see Fig. 1b). Like other members of the hesionidpolychaete family, which live commensally with otherpolychaetes, crustaceans, sipunculans or echinoderms, H. bergi  is associated with the thermophilic annelids Alvinella spp., (i.e. A. condota and A. pompejana , the‘pompeii worm’). It frequently visits tubes of Alvinella spp. for short periods (fewminutes) until ejection bythe occupant worm (see Fig. 2) (Desbruyères et al.1998). H. bergi  is believed to graze on bacteria and/orpredate the small macrofauna associated with thealvinellid tubes, and is therefore likely to encountertemperatures in the same range as those reported forthe habitat of Alvinella species (Desbruyères et al.1982, Chevaldonné et al. 1991, 1992, Cary et al. 1998,Sarradin et al. 1998). By examining the behaviour andsurvival of H. bergi as a function of temperature, weattemped to determine if its survival in the heat of asmoker wall is a matter of biochemical adaptation, inwhich case it would survive and remain unaffected atsustained high temperature in the 50 to 60°C range, asreported in situ by Cary et al. (1998) for A. pompejana .Alternatively, attempts to escape the heat, followed bydeath at lower temperatures would indicate an im-portant role of behavioural responses. MATERIALS AND METHODS In situ  video-observations. During Dive 1382 (of thesubmersible ‘Nautile’ during the French cruise ‘HOPE99’ [NO ‘Atalante’]), almost 1 h of continuous recordingwas obtained over several areas of an alvi-nellidassemblage on a smoker wall (Marker PP 55,12°49.844’N,103°56.812’W). During recording, carewas taken to maintain the imaging-frame on a givenpart of the tube masses, in order to document thebehaviour of the species living there ( Alvinella spp.and other polychaetes, amphipods and peltospiridgastropods). Imaging magnification was as in Fig. 2,sufficient to allow good identification, at the specieslevel, of small fauna such as Hesiolyra bergi  . Pressurised incubator IPOCAMP™. (AutoclaveFrance). The stainless steel vessel (pv) had a volume ofca 19 l. The general design of the pressure circuit wasinspired by the flow-through pressure systems used byQuetin & Childress (1980), with flow rates that reached20 l h –1 at 260 bar working pressure. Pressure oscilla-tions arising from pump strokes (100 rpm) were lessthan 1 bar at working pressure. The temperature of theflowing seawater (through a 0.4 µm filter) was mea-sured constantly, at pressure, in the inlet and outletlines (±1°C). Temperature regulation was poweredbya regulation unit (Huber CC 240) that circulatedethylene-glycol through steel jackets surroundingthepv and through the seawater inlet line. IPOCAMPallowed video-observations of the re-pressurized or-ganisms by combining an endoscope (Fort) to aCCD(charge-coupled device) colour camera (JVC,TK-C1380) (Fig. 1). The results were displayed on aTVmonitor (JVC), and recorded (Sony SVO-9500MDP videotape-recorder). Sample collection and experiments. Hesiolyra bergi  specimens were collected from the East Pacific Riseinthe 13°N hydrothermal vent field at about 2600 mdepth. Collection was achieved with the submersible’shydraulic arm, by ‘claw-grabbing’ among alvinellidcolonies. The animals thus sampled were placed inthetemperature-insulated basket of the submersible.Aboard the NO ‘Atalante’ they were transferred to a14°C cold-room, and H. bergi  specimens were sortedfrom amongst the alvinellid tubes and placed in nylon-meshed cages inside the pv (Fig. 1) at an initial sea-water temperature of 15°C. The cages were closed witha transparent polyethylene lid, secured by nylon line.Upon introduction of the worms, the temperature roseby 1 to 2°C (see temperature curves in Fig. 3a,b), beforebeing cooled to 15°C by temperature-regulating set-tings. To achieve accurate temperature measurements,an autonomous probe (MICREL instruments, ±0.2°C)was placed next to the cages. Re-pressurization at260bar was achieved in about 2 min. In all experiments(corresponding to 4 different collection dives), less than2 h intervened between the time the samples com-menced decompression (submersible ascent) and themoment they were re-pressurized. 142  Shillito et al.: Heat resistance of deep-sea vent annelids The 4 experiments at in situ pressure were of 2 maintypes: (1) maintenance at 15°C (2 experiments, num-bered 1, 2, representing a total of 20 individuals) toverify whether the worms recovered from the collec-tion trauma (‘control’ experiment); (2) heat exposureafter 2 h wait at 15°C (2 experiments, numbered 3, 4,representing a total of 30 individuals). Expt 1:  Eight worms were placed in 1 cage to studybehaviour and survival. In addition, 5 more individualswere placed in sealed containers to evaluate oxygenconsumption (these 5 latter worms could not be ob-served during the experiment). Expt 2:  To reach a total of 15 worms for the survivalstudy at 15°C, 7 more worms were placed in 1 cage tostudy behaviour and survival.In these control experiments, pressure was releasedfor about 15 min (about 2 bar s –1 decompression rate), 6hafter beginning the experiment to allow retrieval of the5individuals (Expt 1: oxygen consumption), and of otherorganisms (Expt 2) that had also been kept in sealed con-tainers in the pv, for other experimental purposes. Expt 3:  Fifteen individuals were placed in the pv in 2cages (7 and 8 individuals, Fig. 1b) and heat-exposedafter 2 h at 15°C until the temperature reached 50°C,followed by cooling to the srcinal 15°C. Expt 4:  Having estimated from Expt 3 that the maxi-mum tolerable temperature was above 40°C, we re-peated the heat-exposure experiment (15 individualsin 2 cages: 7and 8 individuals, respectively), this timeto a temperature of 39°C, i.e. below the previous esti-mation. Cooling back to the srcinal 15°C took place6.5 h after the initial heating.For the heat-exposure experiments (Expts 3 and 4),observations were recorded over 3 periods: during thefirst minutes after re-pressurization; during the heatingand subsequent cooling periods; at the end of the 18 hperiod. As for the controls, this procedure allowed to usdetermine survival at in situ pressure after 18 h, and tostudy the worms’ behaviour during the periods of tem-perature variation (see Fig. 3). Survival after 18 h (all experiments). Survival of there-pressurized worms was determined during the finalminutes of the pressure experiments, by identifyingeach individual and witnessing its movements. Behaviour of worms during heat-exposure vs con-trol experiments (Expts 1, 3, and 4: see Fig. 3). Wormswere individually followed during periods of 30 s atdifferent times during the experiments. Within eachperiod, they were classified into 4 categories:(1) Motionless, no movement detected at normaltape-reading speed; this category was also appliedwhen an individual’s movement seemed to be the re-sult of neighbouring worms ‘pushing’, with no appar-ent reaction of the individual. (2) Any kind of detect- 143Fig. 1. Experimental set-up for heat-exposure experiments. (a) Pressure vessel (pv) contains 2 cages (c) resting on a plastic sup-port (d). Each cage is a cylinder (6 cm diam, 16 cm height) composed of 1 mm-mesh plankton net, closed at the top with a trans-parent polyethylene lid. Extremity of an autonomous temperature probe (h) is located between the cages, about 5 cm above ‘d’,and about 2 cm away from the cage walls. Three connections (x) in the lid of the pressure vessel are terminated by sapphirewindows (w). By inserting an endoscope (ec) into the appropriate connection, content of 1 of the cages may be observed; field ofview is such that the edges of the top of the cage are not visible (dark areas). Illumination is achieved through the other 2 connec-tions by means of optical-fibre light-guides. Large arrows indicate inlet and outlet of circulating seawater. (Scale: internal dia-meter of pv = 20 cm, height = ~60 cm). (b) Video-view of nylon-meshed cage containing 8 Hesiolyra bergi specimens, maintainedin the IPOCAMP at in situ pressure (260 bar) and temperature of 15°C. Parapods of these worms are visible, and appearwhitishdue to the presence of associated bacteria (scale: diameter of cage bottom = ~6 cm, length of worm in centre = ~3 cm) a b  Mar Ecol Prog Ser 216: 141–149, 2001 able movement, at normal tape-reading speed, exceptthat of Category 3 (below): parapod movements (un-dulations), head- or tail-bending, retraction or stretch-ing of body length, forward or backward crawling.(3)Active crawling of the worm (or swimming with nocontact with the substratum), i.e. when it moved alonga distance exceeding its own length in less than 30 s.(4) Worm outside of camera field: this situation corre-sponds to a worm that is either at the top of the cagesor around the edges (see dark areas in Fig. 1a) andtherefore cannot be evaluated, or has escaped the cage(see Fig. 3a,b). If a worm was in the field of the camerabut invisible because other individuals were cover-ingit, it was classed as motionless (Category 1), afterchecking its presence by watching the tape before andafter the 30 s observation period. Oxygen-level measurements (included in Expt 1). Worms were individually stored in soft polyethylenecontainers (60 or 150 ml vol.), that were sealed beforepressurization. Another seawater container (60 ml)without worms was also pressurised for use as a con-trol. After 6 h, these containers were recovered andoxygen levels determined by the Winkler method(SDof the method = 2%; 95% CI for n = 1 is ±4%:Aminot & Chaussepied 1983). The O 2 uptake rateswere checked against the pressurized control to pre-clude possible uptake from bacteria in the seawater.The worms were then dried at 80°C (48 h) andweighed (0.1 mg precision). Electron microscopy (Expts 2 and 4). The heads andtails of the worms were dissected and successivelyfixed in sodium cacodylate-buffered solutions (pH 7.4,4°C): 3% glutaraldehyde (1 h at 4°C), followed by 1%OsO 4 (45 min at 4°C) and subsequent embedding inAraldite resin. Thin sections were stained with uranylacetate and lead citrate and observed with a Leo EM912 Omega electron microscope operating at 120 kV.Micrographs were recorded on Kodak SO 163 film. RESULTS In situ video observations The video-sequence of Dive 1382, which focused ona group of alvinellid tube openings (Fig. 2), was care-fully examined. In this area, we identified at least 7tube openings, of which 2 were definitely occupied by Alvinella spp. individuals.Throughout the sequence, 5 Hesiolyra bergi  speci-mens were visible (as many as 4 simultaneously) amongthe alvinellid tube masses. These worms were fre-quently observed entering and exiting tubes (6 ofthe7tubes), including those occupied by Alvinella spp. Some alvinellids ejected the worms after a fewseconds, but others seemed to tolerate the intruder. H.bergi  sometimes only partially occupied a tube: 3 indi-viduals disappeared entirely inside the tubes for atleast 2 min, whereas another remained for 10 min withabout three-quarters of its body length (posterior part)inside the tube. The worms were also observed wan-dering (over a distance exceeding their body length)over the tube masses. During these wanderings, wormswould sometimes rapidly retract the head upon en-counter with other organisms ( Alvinella spp. smallpardaliscid amphipods and also other H. bergi  spe-cimens). Such ‘avoidance’ behaviour did not alwaysaccompany such encounters. As an alternative to wan-dering, the worms would remain motionless on thesubstratum, eventually stretching, retracting, or bend-ing their body away from the other organisms. Inthevideo-sequences, wenever observed either actualswimming above the substratum or the uncoordinatedbehaviour (spasms) witnessed during the heat-expo-sure experiments. Control experiments Unlike Alvinella spp., Hesiolyra bergi withstood thecollection trauma and could be kept in pressurized sys-tems for at least 48 h. In a preliminary experiment (notdescribed here), 2 individuals survived for more than2d at 260 bar, before the experiment was broken off. 144Fig. 2. In situ view of smoker wall at PP 55 marker site (13°N,EPR), recorded by camera of the submersible ‘Nautile’. Someparts of the image appear blurred due to the mixing of warmfluids with cold seawater. Two Hesiolyra bergi  specimens arevisible (arrows), 1 of them resting above the entrance of anoccupied alvinellid tube (the alvinellid has its red branchialtentacles deployed). They are easily identifiable by 2 whitishlateral lines along their body (bacteria associated with theirparapods). (Scale: diameter of tube occupied by alvinellid = 1 to 2 cm)  Shillito et al.: Heat resistance of deep-sea vent annelids At atmospheric pressure, just after submersible re-covery, or when the pressure was released to allowretrieval of some individuals (oxygen uptake experi-ments), Hesiolyra bergi appeared to be motionless.Only upon careful examination under a dissectingmicroscope could faint parapod or feeding tentaclemovements be detected. However, following re-pres-surization, after only a few minutes (sometimes sec-onds) movements were observed. Detectable move-ments were parapod undulations, lateral flexion ofhead or tail, retraction or stretching of body length,forward or backward crawling. Conversely, whenpressure was released, the worms seemed to instanta-neously lose their ability to move, as evidenced bytheir apparent paralysis at atmospheric pressure.At 15°C, all 15 worms were alive after 18 h: at thistime and at all other times during the experiment, nomore than 5 min continuous observation were neces-sary to detect movements of any individual. Openingof the pressure vessel after 6 h in order to retrieve someworms, (see ‘Materials and methods’) obviously had noeffect on the mortality of the remaining 15specimens,since all survived the 18 h experiment. Throughout the18 h, however, activity remained relatively low: wormswere rarely observed moving more than their ownbody length within a 30 s time period (Fig. 3c). More-over, they remained at the bottom of thecage most ofthe time, and were never observed swimming.In the respirometry experiment, oxygen consump-tion rates correlated well with dry weight (DW) (Fig. 4),to a power of 0.74: (oxygen uptake rate [µl h –1 ]) =2.15·(DW [mg]) 0.74 ; R = 0.982, n = 5, p = 0.003; i.e.,weight-specific oxygen consumption rates for Hesio-lyra bergi  ranged from 630 to 1130 µl g –1 DW h –1 (mean±SD = 895 µl h –1 DW h –1 ±190). The oxygen concen-tration was 245 ±10 µmol in the control after 6 h,whereas the lowest measured concentration for occu-pied containers was 137 ±6 µmol, representing morethan 50% of the initial concentration. Heat-exposure experiments Worms submitted to the first heat-exposure experi-ment (Expt 3: max. 50°C) were all dead after 18 h.Moreover, they had stopped moving during the fourthhour of the experiment, before the temperature hadreached its 50°C max., as evidenced by comparisonofthe relative positions of individuals on all video-sequences after that time until the end of the experi-ment (a slight homogeneous drift [1 to 2 mm] occurredfor all worms, probably due to ship movement). Theseworms were also found to be very fragile, breakingapart (almost dissolving) upon retrieval at the end ofthe experiment. To obtain a more detailed analysis of 145Fig. 3. Hesiolyra bergi. Behaviour during 30 s sequences, as afunction of time since re-pressurization: S , +, H : ‘motionless’,‘moving’, and ‘active crawling’ categories, respectively (see‘Materials and methods’). (a, b) Behaviour (lower graphs) re-lated to temperature (upper graphs, MICREL probe), during 2heat-exposure experiments (Expts 3 & 4, 15 individuals each,split into 2 groups of 7 and 8 individuals). Endoscope wasmoved from one cage to the other (see Fig. 1a), and behav-ioural data for the last 30 s in one cage and the first 30 s in theother cage were pooled; because of possible delay in endo-scope adjustments, 1 to 3 min may separate a pair of se-quences. For each observation time, the sum of the 3 behav-ioural categories equals the number of observable worms(grey line, showing that at some point in the experiment someworms were outside the camera field or had escaped, i.e. theybelonged to the 4thcategory, ‘worm outside camera view’: see‘Materials and methods’). Maximum heating/cooling rateswere 0.44 and –0.28°C min –1 , respectively, decreasing whenthe temperature neared the target-values of the thermoregu-lating unit. (c) Behaviour of 8 individuals in the same cage at15°C (Expt 1). They were filmed continuously for periods of atleast 25 min, at different times of the 18 h experiment (the lastperiod, 30 min in the 18th hour, is not shown). Within these pe-riods, 30 s sequences were studied approximately every 3min(SD = 25 s), representing 93 sequences. On the graph, onlyevery fourth sequence is represented (every 12 min). Overall,active crawling almost never occurred; it was only observedduring 5 sequences out of a total of 93: 1 individual out of 8(12.5%) in 4sequences; 2 out of 8 (25%) in 1 sequence. Ofthese 5 sequences, 4 took place during the 18th hour. Mean±SD (%) for ‘motionless’, ‘moving’, and ‘active crawling’ cate-gories are 54.4 ±26.2, 44.1 ±25.1, 0.8 ±3.6 (n = 93), respectively
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