Heat-shock response and temperature resistance in the deep-sea vent shrimp Rimicaris exoculata
1 UMR CNRS 7138 `Systématique, Adaptation et Evolution',
Université Pierre et Marie Curie, 7 Quai St-Bernard, Batiment A, 75252
Paris Cedex 05, France
2 Institut Français de Recherche pour l'Exploitation de la Mer
(IFREMER), Centre de Brest, DRO-EP, bp 70, 29280 Plouzané,
France
3 Station Marine de Roscoff, UPR CNRS 9042, Equipe `Evolution et
génétique des populations marines', bp 74, 29682 Roscoff Cedex,
France
* Author for correspondence (e-mail: bruce.shillito{at}snv.jussieu.fr)
Accepted 31 March 2003
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Summary |
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Key words: hydrothermal vent, thermal stress, Crustacea, Caridae, IPOCAMP, chaperone, Rimicaris exoculata, shrimp, heat shock
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Introduction |
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The exposure of organisms to elevated temperature induces the expression of
heat-shock proteins (hsp; Lindquist,
1986). In most organisms studied so far, hsp70 proteins are among
the most prominent proteins induced by heat, and these proteins do play a
central role in tolerance to high temperatures, as they allow cell survival
during and after thermal stress (reviewed in
Parsell and Lindquist, 1993
).
Most research on these proteins has focused on cellular and molecular aspects,
but there is a growing interest in approaching the organismal, ecological and
evolutionary aspects of the stress response (reviewed by
Feder and Hofmann, 1999
;
Queitsch et al., 2002
).
Because R. exoculata lives in a complex and highly fluctuating
thermal environment and is thought to undergo frequent harsh temperature
conditions on the chimney wall, it provides a useful biological system for
stress-response studies.
The central questions underlining this work are: what is the absolute upper
thermal limit tolerated by the host shrimp and, assuming that temperatures
above 20°C are optimal for the epibionts
(Wirsen et al., 1993), are
exposures to such temperatures stressful for their hosts? To address these
questions, we undertook several experiments with live shrimps maintained in
video-equipped pressurized aquaria. Our goal was first to determine the upper
thermal tolerance (CTmax) of the shrimp by following
behavior and survival upon severe heat shock (45°C peak). Second, we
investigated some characteristics of the stress response (heat-shock protein
accumulation) of R. exoculata during a `mild' heat shock (25°C
peak) that would presumably be biologically and environmentally relevant. In
parallel, reference experiments (survival, behavior and oxygen consumption)
were conducted at 15°C.
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Materials and methods |
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Pressurized incubator IPOCAMPTM
The stainless steel IPOCAMP (Incubateur Pressurisé pour
l'Observation et la Culture d'Animaux Marins Profonds) pressure vessel (PV)
has a volume of approximately 19 liters, as previously described
(Shillito et al., 2001). The
general design of the pressure circuit is inspired from flow-through pressure
systems utilized by Quetin and Childress
(1980
), with flow rates that
may exceed 20 liters h-1 at 230x105 Pa working
pressure. Pressure oscillations due to pump strokes (100 r.p.m.) are less than
1x105 Pa at working pressure. The temperature of the flowing
seawater (filtered at 0.4 µm) is measured constantly, at pressure, in the
inlet and outlet lines (±1°C). A more accurate temperature
measurement (±0.1°C) is achieved inside the pressure vessel through
two Pt-100 probes (see Fig. 1).
Temperature regulation is powered by a regulation unit (Huber CC 240) that
circulates ethylene glycol around the seawater inlet line and through steel
jackets that surround the PV. Finally, IPOCAMP allows video observations of
the re-pressurized organisms by combining an endoscope (Fort, Dourdan, France)
to a CCD camera (JVC, TK-C1380; Fig.
1). The resulting view is further displayed on a TV monitor (JVC)
and recorded (Sony SVO-9500 MDP videotape recorder).
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In vivo experiments
Once on board, R. exoculata specimens were transferred to a
14°C cold-room. Most shrimps survived the collection trauma, and the
individuals that swam apparently normally were sorted out to be re-pressurized
for in vivo experiments. Only adult specimens were chosen, and these
specimens were placed in PVC cages inside the pressure vessel at an initial
seawater temperature of 15°C, as previously described by Shillito et al.,
(2001). Re-pressurization at
230x105 Pa was achieved in approximately 2 min. In all
experiments, less than 2 hintervened between the time the samples began
decompression (submersible ascent) and the moment they were
re-pressurized.
Four experiments at in situ pressure (230x105 Pa; 20 liters h-1 flow rate) were performed on a total of 60 shrimps. For each experiment involving behavioral observations (all but Expt. 1), 15 shrimps were placed in three cages (five individuals per cage; Fig. 1B).
Expt 1 (respirometry experiments A and B, at reference
temperature)
The shrimps were placed for 3 h (Expt 1A) or 4 h (Expt 1B) at 15°C in
order to evaluate their oxygen consumption. These animals were not analyzed
for their behavior. Because these experiments were performed in closed
containers (see Oxygen-level measurements), the oxygen level decreased as a
function of time, which limited the experiment duration to 4 h.
Expt 2 (reference experiment)
The shrimps were maintained at 15°C over 24 h. This `control'
experiment aimed to study behavior and survival in order to verify whether the
shrimps recovered from the collection trauma. These shrimps also served as a
control for immunochemical analysis (see below).
Expt 3 (lethal heat shock)
After 5 h at 15°C, the shrimps were heat-exposed, as previously
described for the vent polychaete Hesiolyra bergi
(Shillito et al., 2001), until
the temperature reached 45°C, followed by cooling to 15°C. Maximum
heating and cooling rates were 0.52°C min-1 and -1.05°C
min-1, respectively. The total duration of the experiment was 8
h.
Expt 4 (mild heat shock)
From the results of Expt 3, we tried a milder heat-exposure experiment to a
temperature of 25°C. The heating phase started 21 h 45 min after the
re-pressurisation, and the cooling phase to 15°C took place approximately
1 h later. Maximum heating and cooling rates were 0.35°C min and
-0.29°C min-1, respectively. The total duration of the
experiment was 24 h, as for Expt 1 (control).
All the shrimps experimented on during Expt 2 and Expt 4 were frozen in liquid nitrogen on board until further analyses at the lab.
Video analysis of in vivo experiments
For all the in vivo experiments, survival of the re-pressurized
shrimps was determined during the last minutes of the experiments by
identifying each individual and witnessing its movements. Additionally,
survival could also be confirmed at atmospheric pressure after the
experiments.
For the heat-exposure experiments (Expts 3 and 4), observations were recorded throughout the experiment.
The endoscope was moved successively from the first to the third cage (4 min observation period for each cage) about once every hour at 15°C and then continuously rotated during heat shock (each cage was observed for 4 min before moving to the next). The resulting behavioral data for 15 shrimps were pooled from the last 30 s in the first cage, the middle 30 s in the second cage, and the first 30 s in the third cage (see also Fig. 2 legend). Within each period of observation, the shrimps were individually classified into four categories:
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(C1) `motionless': no movement detected at normal tape-reading speed; this category was also applied when an individual's movement seemed to be the result of neighboring shrimp `pushing', with no apparent reaction of the individual.
(C2) `moving': any kind of detectable movement, at normal tape-reading speed, except that of category 3 and 4 (below): pereiopod or pleopod movements, scaphognathite beating, antennal lateral sweeping on dorsal side, or cleaning of the mouth parts by rubbing them along each other.
(C3) `active walking or swimming': when the shrimp moved along a distance exceeding its own length in less than 30 s.
(C4) `spasms': spasmodic motions (vibrations of the abdomen) without any
coordinated movement; the temperature at which the animals were first observed
to move in a jerky uncoordinated way was defined as the critical thermal
maximum (CTmax; Wehner
et al., 1992; Gehring and
Wehner, 1995
; Cuculescu et al.,
1998
).
All the categories are exclusive; i.e. one individual can only be assigned once among these four categories.
For each observation point of 15 individuals during heating periods (successive sequences on the three cages), we determined the error limits for the corresponding temperature. Maximum heating rates were 0.52°C min-1 (Fig. 2A) and 0.35°C min-1 (Fig. 2B) for Expts 3 and 4, respectively. Considering the maximum heating rates and the duration of each video survey (5 min, from the last 30 s of the first sequence, through the middle 4 min of the second sequence, to the first 30 s of the third sequence), a resulting maximum variation of 2.6°C (Expt 3) and 1.7°C (Expt 4) in the temperature measurements was obtained. Moreover, during heating, a maximum difference of 1.7°C was recorded between the two temperature probes placed at the top and the bottom of the cage. For each temperature point, the maximal error is thus approximately ±2°C (Expt 3) and ±1.7°C (Expt 4).
Oxygen-level measurements
Shrimps (15 individuals; Expt 1) were individually stored in soft
polyethylene containers filled with 210 ml of surface seawater, which were
sealed before pressurisation. Another 210 ml container without any animals was
also pressurized for use as a control. After either 3 h or 4 h (Expt 1A or 1B,
respectively), these containers were recovered and the oxygen concentration
was estimated using a Clark-type microelectrode (DK; Unisense, Aarhus,
Denmark) with an estimated precision of ±3%. These measurements were
calibrated with air-equilibrated surface seawater (100% O2) and
deoxygenated surface seawater by addition of sodium sulphide (0%
O2). The 100% O2 solution was standardised with the
Winkler method (S.D. of the method = 2%; 95% confidence interval
for N=1 is ±4%; Aminot and
Chaussepied, 1983). The O2 uptake rates were checked
against the pressurized control to preclude possible uptake from bacteria in
the seawater. No oxygen consumption was registered in the controls.
The shrimps were dried at 80°C on board the ship (for 48 h). They were
then further dried at the lab at 80°C (for 5 days) and weighed (0.1 mg
precision). Among the 15 shrimps treated, only the individuals for which the
final oxygen concentration in the containers did not drop below 50% of the
initial concentration were kept for the results interpretation (seven
individuals). Final O2 content was thus definitely above the oxygen
level at which the shrimp O2 consumption may decline rapidly
(Prosser, 1973).
Electrophoresis and immunochemical analysis (western blot) of
hsp70
Samples of shrimp abdomens (from Expts 2 and 4) without their cuticle were
ground in liquid nitrogen, and the powder was homogenised in 1.5 ml of
extraction buffer [10 mmol l-1 Tris/HCl, pH 7.4:protease inhibitor
cocktail (Sigma, St Quentin Fallavier, France) 1:3 (v/v)]. The homogenates
were sonicated (2x10 s) using a cell homogeniser and centrifuged at 10
000 g for 10 min at 4°C. The pellet was discarded and the
extracted proteins were quantified in the supernatant with the Bio-Rad Protein
Assay (Bio-Rad, Marnes-la-Coquette, France) using bovine serum albumin (Sigma)
as standard. Proteins of the total supernatant were separated by minigel
sodium dodecyl sulfatepolyacrylamide gel electrophoresis
[SDSPAGE; 10% acrylamide:0.3% bisacrylamide (w/v); 4 µg protein
well-1]. After running the electrophoresis (15 min at 15 mA
gel-1, 1 h at 20 mA gel-1), the bands were stained with
silver nitrate (Wray et al.,
1981). Protein molecular mass standards from 29 kDa to 205 kDa
(Sigma) were used to evaluate the apparent Mr of the
separated bands.
For western blotting, the proteins were transferred from the SDSPAGE gel (20 µg protein well-1) to a nitrocellulose membrane by semi-dry blotting at 20 V for 1.5 h (Trans-Blot semi-dry cell; Bio-Rad). Membranes were blocked in milk/Tris-buffered saline pH 7.4 (TBS) (5% w/v) for 1 h and incubated overnight with a polyclonal antibody (anti-human hsp70; StressGen, Victoria, BC, Canada) at room temperature. Subsequently, a 10 min washing step with TBS pH 7.4 was repeated three times, and the membranes were incubated with a polyclonal secondary antibody coupled to peroxidase (StressGen) for 2 h at room temperature. After another 3x10 min washing step with TBS, the antibody complex was detected using the substrate Bromo-chloro-indolyl-phosphate/Nitro Blue Tetrazolium (BCIP/NBT) (StressGen) incubated at room temperature for 10 min. The membranes were further digitalized using a UMAX (Hsinshu, Taiwan) Power Look 3 scan at 600 d.p.i. resolution. Density profiles were obtained from the western blot membranes using NIH Image 1.6 software.
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Results |
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At 230x105 Pa and 15°C (Expt 2), all the shrimps were alive after 24 h. They can be maintained alive in pressurized vessels (230x105 Pa; 15°C) for at least 48 h with a survival rate of 100% (preliminary experiment with 15 specimens, not described here). A behavioral survey throughout Expt 2 (16 observations of 30 s sequences) allowed the detection of a broad diversity of movements. Table 1 summarizes the behavioral response of the shrimps during the different periods of maintenance at 15°C of Expt 2 (24 h), Expt 3 (first 5 h) and Expt 4 (first 21 h), which represents a total of 33 observations. This survey of periods at 15°C allowed us to set the threshold values for considering significant behavior during the heat-shock experiments. Throughout the 24 h of Expt 2, the shrimps were frequently observed immobile (C1) or slightly moving (C2). Several movements of the different appendages were identified: pereiopod or pleopod movements, antennal lateral sweeping on the dorsal side, and cleaning of the mouth parts (particularly the maxillipeds) by rubbing them along each other. The shrimps rarely swam or walked actively (C3), and a maximum of seven individuals were observed actively swimming at the same time. According to this, the threshold value considered in the heat-shock experiments for activity above the reference level was seven individuals. During these 15°C periods, the shrimps never had spasms (C4). The branchiostegites (lateral sides of the cephalothorax) sometimes appeared transparent, which allowed us to see the scaphognathite (large flaps on the second maxillae) beating in the gill cavities when the animals were in side view or when they faced the endoscope (vertical position on the cage wall; see Fig. 1). The scaphognathite movements create a water flow on the gills, and the beating rate was 2458 beats min-1 (at 15°C; N=12).
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In the respirometry experiment, the initial oxygen content in the flasks roughly corresponded to the oxygen saturation level in seawater at the temperature of the experiments (254±9 µmol l-1; N=5). This level is close to the oxygen content of 240±9 µmol l-1 (N=4) that we determined for deep waters surrounding the hydrothermal vent fields. The oxygen consumption rates (R; expressed in µgO2 h-1) were related to the shrimps mass (M; expressed in mg dry mass) following the equation R=1.748M0.891 (r=0.832, N=7, P<0.05; Expt 1A,B; Fig. 3). The mass-specific oxygen consumption rates for Rimicaris exoculata were 0.8371.094 mg O2 g-1 dry mass h-1 (means ± S.D. = 0.979±0.101 mg O2 g-1 dry mass h-1).
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Heat-exposure experiments
Temperature resistance
Shrimps submitted to the first heat-shock experiment (Expt 3; maximum
temperature 45°C; Fig. 2A)
were all dead after 8 h. Moreover, they had stopped moving during the seventh
hour of the experiment, before the temperature had reached its 45°C
maximum. Indeed, from this point onwards, the shrimps all remained motionless
at the bottom of the cage, laid on their back or side, and finally assumed a
curved body shape in a post mortem position. The influence of
temperature was analyzed by observing the behavior of these shrimps over 18
video-sequences of 30 s throughout the experiment (four behavioral categories;
see Materials and methods; Fig.
2A). During heat exposure, an increase in shrimp activity (C3:
active walking or swimming) above the reference level was observed, which
started between 24±2°C and 28.5±2°C (see legend of
Fig. 2; see
Table 1 for reference
behavior). The peak of this activity response corresponds to approximately
33°C (13 shrimps among the 15 were swimming or actively walking) and was
followed by a fast decrease in activity until 45°C. This decrease was
accompanied by an apparent loss of locomotory coordination, expressed as
flicking of the abdomen (spasms) and rapid movements of the pleopods without
any efficient displacement. All the animals were dead when the temperature
reached 43±2°C.
Heat-shock response
During the second heat-exposure experiment (Expt 4;
Fig. 2B), which was non-lethal,
the maximum temperature was 25°C and all the shrimps survived after 24 h.
The behavior during the first 21 h (15°C; 230x105 Pa) was
similar to that of the reference experiment (Expt 2; see
Table 1). The activity was
relatively more intense during the heat exposure, as more than seven
individuals were frequently observed swimming (C3) at the same time, with a
maximum (12 individuals) at around 25°C. These shrimps were further
analyzed for the detection of stress proteins.
The protein profiles of reference (Expt 2) and heat-shocked (Expt 4) R. exoculata abdomen samples are shown in Fig. 4 (only one profile is presented for each type of sample). In the heat-shocked individual, at least three proteins, with molecular masses of about 205 kDa, 90 kDa and 70 kDa, are overexpressed compared with the reference individual, for which these proteins display very low abundances.
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The relative difference in the level of the hsp70 stress proteins between the groups of four reference (R) and four heat-shocked (HS) R. exoculata in response to a 25°C heat exposure (approximately 15 min at 25°C; see Expt 4 and Fig. 2B) was quantified by western blot and subsequent densitometry analysis using the NIH software (Fig. 5). hsp70 proteins were detected in both groups as two protein bands, one of them being more intense in the heat-shocked individuals [low molecular mass (LMM) band; Fig. 5A]. The intensity of the high molecular mass (HMM) protein band, which was revealed in all samples, is not significantly different between the R and HS samples (MannWhitney test; U=6.0, P=0.564). On the contrary, a significant increase of the LMM protein band intensity is observed on the western blot of the shocked animals (Fig. 5A,B; MannWhitney test, U=0.0, P=0.021).
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Discussion |
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The reference experiments were performed in conditions comparable with
those in situ for pressure (230x105 Pa) and oxygen
level (240 µmol l-1). Our 15°C reference temperature was
chosen in agreement with the mean temperature values recorded so far in the
shrimp microenvironment: 11°C (Geret
et al., 2002), 13.2±5.5°C
(Desbruyères et al.,
2001
) and 1015°C
(Segonzac et al., 1993
). This
reference temperature is significantly lower than the maxima suggested to be
encountered in the shrimp habitat, where usual temperatures may reach,
according to the various authors, 25°C
(Desbruyères et al.,
2000
), 30°C (Van Dover et
al., 1988
) or 40°C (Gebruk
et al., 1993
).
Comparisons of experimental behavior of the shrimp with natural behavior
should be made cautiously, since both environment and observation conditions
differ radically. Moreover, whereas the individuals often live in dense swarms
in situ, only five individuals were used in a single cage for
pressure-vessel experiments. However, the various types of behavior observed
experimentally resemble those occurring in situ, except for the lack
of uncoordinated flicking of the abdomen (spasms). Most of the time, the
shrimps were observed either motionless on the bottom or along the walls of
the cage or making small movements of the appendages. In their natural
environment, the shrimps may also remain motionless on the surface of the
black smoker chimneys, moving only slightly in confined areas
(Van Dover et al., 1988;
Gebruk et al., 1993
,
2000
;
Segonzac et al., 1993
). They
were occasionally observed actively swimming, when entrained by turbulent
water, to move back towards the substrate.
Temperature resistance
In the 45°C peak heat-exposure experiment (Expt 3), none of the 15
shrimps survived. A significant increase in active crawling appeared in the
2428.5°C temperature range, which, in view of the low activity at
15°C, can be inferred to be an escape response to avoid heat zones. A
similar behavior occurs in situ, since the shrimps lay more or less
motionless on chimney walls, where the measured temperature was 2°C,
whereas they swam more actively as they came across warm water zones
(Segonzac et al., 1993).
However, the escape response observed for R. exoculata below
28.5°C is inconsistent with other in situ observations reporting
a slight mobility of individuals in swarms where the temperature was reported
to have reached 30°C (Gebruk et al.,
1993
,
2000
). A more detailed
description of the thermal characteristics of the R. exoculata
microenvironment is necessary to understand the in situ shrimp
behavior and, in particular, it is of interest to determine whether the
shrimps are able to regulate their microhabitat temperature by inducing water
flows with their locomotory appendages.
The critical thermal maximum (CTmax) is defined as the
temperature at which the animal is no longer capable of proper locomotion and
starts to move in a jerky uncoordinated way, i.e. the temperature at which the
first signs of heat stress occur (Wehner
et al., 1992; Gehring and
Wehner, 1995
; Cuculescu et al.,
1998
). CTmax for R. exoculata is
evidenced by very characteristic spasmodic movements of the abdomen and is in
the 3337°C range (±2°C; see
Fig. 2A). The shrimps were all
dead when the rising temperatures reached 43±2°C, as they all sank
to the bottom of the vessel and laid motionless on their backs or sides.
R. exoculata may be compared with another vent crustacean species,
the East Pacific Rise crab Bythograea thermydron, which survives a 1
h exposure at 35°C but succumbs after 1 h at 37.5°C
(Mickel and Childress, 1982
).
The CTmax value for R. exoculata is lower than
that of the vent polychaete H. bergi (4146°C; East Pacific
Rise), which lives on the chimney walls and is likely to encounter occasional
high-temperature pulses (Shillito et al.,
2001
), and would also be very different from that proposed for the
vent annelid Alvinella pompejana (East Pacific Rise), which,
according to Cary et al., would lie above 60°C at least
(1998
). The upper thermal
tolerance of R. exoculata is close to that of the Iceland hot spring
beachflea Orchestia gammarellus (approximately 37°C; microhabitat
temperatures ranging from 15°C to 25°C; Bjarnarstadir site;
Morritt and Ingolfsson, 2000
),
which occupies similar harsh and unstable environments where temperatures can
reach 42°C (thermal spring temperature). Finally,
CTmax for R. exoculata is also comparable with
that of the shore crab Carcinus maenas (approximately
3234°C for 15°C-acclimated specimens;
Cuculescu et al., 1998
), which
is regularly exposed to severe abiotic stresses and experiences very high and
rapid changes in body temperature during the tidal cycles.
Stress response/stress protein induction
The heat-shock response is universal, as almost all organims studied to
date are able to express heat-shock proteins (hsp; reviewed in
Lindquist, 1986). Lindquist
(1986
) suggested that only
some creatures living in the depths of the ocean may not have a heat-shock
response, but even this was doubtful. This study constitutes the first
analysis of the heat-shock response for a deep-sea vent organism living at
2300 m depth in a highly fluctuating thermal habitat that should cause
frequent heat stress.
At least two proteins of the hsp70 family are present in both reference and
heat-shocked animals. However, one of them (LMM band; see
Fig. 5) was clearly more
abundant in animals that were submitted to a heat shock, thus demonstrating
its involvement in the heat-shock response. According to this, the presence of
a less intense LMM band in reference animals could possibly indicate that
15°C is a temperature that approaches the hsp70 induction threshold.
Alternatively, it may reflect a `background' response to experimental stress
(pressure variations upon recovery and conditioning in IPOCAMP, manipulation,
etc.), since hsp70 expression can be triggered by many non-thermal stresses
(Feder and Hofmann, 1999),
including pressure variations (Welch et
al., 1993
). Finally, this low-intensity band may signal natural
heat stress prior to sampling, even though the shrimps had been collected
in situ at least 10 h before they were frozen for hsp detection.
Indeed, it has been shown in the case of marine snails
(Tomanek and Somero, 1999
)
that the presence of heat-inducible hsp70 was still detectable almost 50 h
after an initial heat shock.
The HMM band was equally intense in both reference and heat-shocked animals (Fig. 5) and therefore appears to reflect a non-heat-inducible form of the hsp70 family. This protein could be either a constitutive form of the hsp70 chaperone [i.e. the so-called heat-shock cognates (hsc), which are expressed continuously in the organism] or could again be triggered by the experimental stress. None of the previous hypotheses can be favored but, because about the same level of this hsp70-like protein was found in all eight experimental animals, the HMM-band protein does not seem to be involved in the heat-stress response, whereas the LMM-band hsp70 form definitely is.
Under elevated thermal conditions, the hsp70 proteins function as
`molecular chaperones', preventing the aggregation and promoting the proper
refolding of denatured proteins (reviewed in
Parsell and Lindquist, 1993).
In R. exoculata, the synthesis of an hsp70 heat-inducible form
occurred following a heat exposure at 25°C (approximately 15 min at
25°C; Expt 4; Fig. 2B) and
thus may reflect the emergence of cellular damage in the shrimps. The
threshold induction temperature of the heat-shock response (i.e. the
temperature at which heat-inducible hsp isoform synthesis is first observable)
is thus lower than 25°C. The hsp70 enhanced synthesis threshold in R.
exoculata would be in the same range of temperatures as for the
13°C-acclimated marine snail Tegula brunnea (24°C;
Tomanek and Somero, 1999
) or
for various 10°C-acclimated teleost fishes (2028°C range;
Dietz and Somero, 1993
).
However, the hsp70 expression in R. exoculata occurred after a
relatively brief heat exposure (15 min at 25°C) when compared with the
heat exposure of the marine snail (2.5 h;
Tomanek and Somero, 1999
) and
the teleost fishes (2 h; Dietz and Somero,
1993
). As the induction of hsp reflects stress conditions for the
organism (Parsell and Lindquist,
1993
), the optimal habitat temperature range of R.
exoculata may be below 25°C. This is quite low considering that the
shrimps should approach the hydrothermal fluid to `farm' their epibionts and
are thought to tolerate in situ temperature spikes of at least
40°C (Gebruk et al.,
1993
). Furthermore, juveniles of R. exoculata have been
observed in zones of diffuse vent fluid where the temperature is above
20°C (Gebruk et al.,
2000
), and adult shrimps are supposed to live at temperatures of
up to 30°C (Van Dover et al.,
1988
; Gebruk et al.,
1993
). Several questions arise when considering the definition of
the habitat temperature range of these shrimps. In such a highly fluctuating
environment, the relevance of maximum temperature obtained from discrete
measurement should still be considered cautiously. Furthermore, the shrimps
can reach densities of 3000 individuals m-2 in the swarms
(Gebruk et al., 2000
), where
they are packed side by side and often piled two or more deep (up to 0.4 m
thickness), which can interfere with the probing effort.
This study reports the first in vivo experiments in pressurized aquaria on a deep Mid-Atlantic Ridge vent organism. The heat-shock experiments with a protein induction assessment provide an indication of the thermal limits of this species, suggesting that the habitat temperature would be restricted to values lower than previously expected. The conclusion that can be drawn from our experiments is that R. exoculata does not tolerate exposure to temperatures in the 3337°C range (CTmax) and succumbs to heat stress above this limit. Moreover, the shrimps synthesize heat-shock proteins when briefly exposed (15 min) to a temperature of 25°C. R. exoculata would thus be forced to live in a narrow thermal window in which the lower and upper limits are set by the epibionts `farming' demands and heat stress, respectively, and would occasionally be exposed to high temperature peaks during very short periods. Considering the great difference in volume between the shrimps and their bacteria, they certainly undergo very different internal temperature patterns for the same environmental temperature conditions. The optimal thermal window or thermal regime allowing a successful epibiosis has still to be determined.
Further studies will help to pinpoint the threshold temperature for the
induction of enhanced hsp70 expression in the shrimp. Since inducible hsp70 is
thought to be deleterious when expressed under non-stress conditions (reviewed
by Parsell and Lindquist,
1993; Krebs and Feder,
1997
), the heat-shock response needs to be transient, which would
imply for R. exoculata the ability to rapidly express and inactivate
the hsp70. Future research will aim to determine whether adaptation to the
fluctuating hydrothermal environment is reflected by particular kinetics of
the hsp response in the vent shrimps.
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Acknowledgments |
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References |
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