|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online June 27, 2008
Journal of Experimental Biology 211, 2196-2204 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018606
Thermal biology of the deep-sea vent annelid Paralvinella grasslei: in vivo studies
1 UPMC Université Paris 6, UMR 7138, `Systématique, Adaptation et
Evolution', F-75005 Paris, France
2 CNRS UMR 7138, `Systématique, Adaptation et Evolution', F-75005, Paris,
France
3 UPMC Université Paris 6, FRE 2852 `Protéines: Biochimie
Structurale et Fonctionnelle', F-75005 Paris, France
4 CNRS FRE 2852 `Protéines: Biochimie Structurale et Fonctionnelle',
F-75005 Paris, France
5 DEEP/Laboratoire Environnement Profond, Centre IFREMER de Brest, bp 70, 29280
Plouzané, Cedex, France
* Author for correspondence (e-mail: juliette.ravaux{at}snv.jussieu.fr)
Accepted 1 May 2008
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Key words: Hydrothermal vents, heat shock proteins, stress response, heat stress, annelids, IPOCAMP
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
). As a
consequence, the vent fauna have to deal with harsh and highly unstable
thermal conditions. In the last few years, several studies have reported that
some vent creatures, and in particular annelids belonging to the family
Alvinellidae, feature exceptional thermal characteristics. This is the case
for the emblematic polychaetous annelid Alvinella pompejana, believed
to be one of Earth's most temperature-resistant metazoans, as suggested by the
thermal stability of the molecules comprising its extracellular matrix (Gaill
et al., 1993; Gaill et al.,
1995). In situ recordings have suggested that A.
pompejana could survive sustained temperatures of up to 60°C
(Cary et al., 1998) and brief
exposures beyond 100°C
(Chevaldonné et al.,
1992). Unfortunately, despite recent encouraging results
(Shillito et al., 2004), the
trauma associated with deep-sea collection has not yet allowed determination
in vivo of the upper thermal limits of this species, an issue that
remains controversial (Chevaldonné
et al., 2000). Conversely, however, another member of the same
family, Paralvinella sulfincola, which displays a similar association
with hot fluid emissions (Juniper et al.,
1992), survives throughout deep-sea collection, and was recently
studied in vivo inside pressurized aquaria
(Girguis and Lee, 2006). It
was found to tolerate temperatures of 50–55°C for several hours, the
highest temperature ever found for a marine metazoan. This worm species
apparently preferred temperatures between 40 and 50°C. Beyond establishing
the upper thermal limits of these peculiar congeners by using exposure at
close to lethal temperatures, another way to explore the thermal biology of
these animals is to seek the first signs of thermal stress, through `mild'
heat shocks, i.e. thermal exposures that are possibly stressful, but are not
of long enough duration to be lethal. This approach may provide complementary
data regarding the capacity of vent fauna to get close to vent emissions. This
ability could provide a selective advantage in interspecies competition
(Tunnicliffe, 1991), which is
particularly relevant in this thermally oscillating habitat, where a temporary
access to hotter areas may provide a refuge from predators and/or access to
specific nutritional sources.
In this preliminary study, we firstly followed the behaviour and survival
of the vent-endemic alvinellid worm Paralvinella grasslei during a 30
min heat shock at 
30°C in a video-equipped pressurized aquarium.
Secondly, we investigated its stress response measured by heat shock protein
accumulation after the heat shock. When organisms are exposed to a non-lethal
thermal stress, the expression of a highly conserved set of polypeptides
termed heat shock proteins (HSPs) is initiated
(Feder and Hofmann, 1999).
These proteins play an essential role in the repair or destruction of damaged
proteins (Parsell and Lindquist,
1993). In most organisms studied, the most prominent proteins
induced by heat stress are HSP70 proteins (so called because their molecular
mass is approximately 70kDa) (Feder and
Hofmann, 1999).
P. grasslei is one of the most abundant polychaete species found
at the East Pacific Rise (EPR) vents. It occurs both within A.
Pompejana colonies, on the wall of active chimneys, and within Riftia
pachyptila tube aggregations
(Desbruyères et al.,
2006). Therefore, this species may undergo temperature conditions
as low as 10°C [in Riftia pachyptila tubeworm clumps
(Sarradin et al., 1998)],
while it may also be exposed to temperatures well above 30°C [among A.
pompejana colonies (Le Bris et al.,
2005)]. Given the relative mobility of this species
(Chevaldonné et al.,
2000), could this rather wide thermal distribution reflect
frequent displacements between different thermal environments, as has been
suggested for the vent shrimp Rimicaris exoculata
(Ravaux et al., 2003), rather
than a static distribution of fixed sub-groups, each adapted to different
thermal regimes? By using video observations during in vivo heat
exposure experiments at in situ pressure, and postshock detection of
stress proteins, we aimed to demonstrate both the feasibility and scientific
contribution of such shipboard physiological ecology studies. Finally, by
presenting stress protein sequences of members of the Alvinellidae, this work
provides valuable investigation tools for future studies on the thermal
biology of this family of annelids.
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
) specimens were collected during two cruises: `HOPE' (R/V
Atalante, ROV Victor6000, April 1999) and `PHARE' (R/V Atalante, Nautile
submersible, June 2002) along the EPR at the 13°N hydrothermal vent field
at about 2600 m depth. Animals were sampled with a suction device operated by
the submersible's hydraulic arm, and stored inside insulated Perspex cylinders
until transferred to the ship. Most annelids survived the collection trauma,
and only live adult specimens (4–7 cm in length) were placed in PVC
cages inside the IPOCAMP pressurized incubator for in vivo
experiments (Fig. 1) (for
details, see Ravaux et al.,
2003). In all experiments, less than 2 h passed between the time
the samples began decompression (submersible ascent) and the moment they were
re-pressurized. After experimentation, live worms for further analyses at the
lab were immediately frozen in liquid nitrogen.
|
|
Respirometry experiment (Expt 1)
Five worms were placed at 15°C for 6 h in order to estimate their
oxygen consumption. This experiment aimed at evaluating the physiological
state of the annelids after the collection process. These animals could not be
observed, as this experiment was performed in individual closed
containers.
Reference experiments (Expts 2 and 3)
Five and six P. grasslei were maintained at 15°C for 9 h (Expt
2) and 6 h (Expt 3), respectively. The aim of these experiments was to study
behaviour and survival in order to determine whether the worms recovered from
the collection trauma.
Heat shock experiments (Expts 4–6; see Table 1)
We performed three non-lethal heat shock experiments at 30°C with a
similar temperature profile (Fig.
1B) but with different recovery periods. After being maintained at
15°C for 8 h, the worms were exposed to an abrupt heat shock. The
temperature of the in-flowing seawater was increased in less than 5 min by
immersing the inlet high-pressure tube in a 30°C regulated water bath
while directing this flow into the experimental cages (see
Fig. 1A). Animals were
maintained for at least 30 min, followed by a rapid cooling to the original
15°C (within about 5 min). Animals were taken out of the chambers and
frozen immediately (Expt4a), or frozen 1.5h (Expt5a) or 3.5 h (Expt 6a) after
the heat shock for further investigations on heat shock proteins. For each
heating experiment, a reference experiment of the same total duration was
conducted simultaneously, at a constant temperature of 15°C and at in
situ pressure, using a second IPOCAMP pressure vessel (Expts 4b, 5b and
6b). All of the heating experiments were video monitored, in order to detect
any type of behavioural response to thermal stress.
Oxygen level measurements (Expt 1)
Worms were individually sealed in soft polyethylene seawater containers
(60, 125 or 150 ml) in order to evaluate their oxygen consumption. Another
seawater container without any animals was pressurized as a control. After 6
h, all containers were recovered and oxygen levels determined by the Winkler
method (s.d. of the method was 2%; 95% confidence interval for N=1
was ±4%) (Aminot and Chaussepied,
1983). The O2 uptake rates were compared with the
control to preclude possible uptake of oxygen by bacteria in the seawater. The
worms were then dried at 80°C (48 h) and weighed (0.1 mg precision).
Determination of survival and video analysis of in vivo experiments
For all in vivo experiments with a video survey, survival of each
individual re-pressurized worm was determined by witnessing its movements
during the last 5 min of the experiments. Survival of each specimen was
confirmed after the experiments at atmospheric pressure by identifying any
kind of movement of the animal.
The pressure vessel IPOCAMP allows video observation of the animals by
combining an endoscope (Fort, Dourdan, France) with a CCD camera (JVC,
TK-C1380; Carrières sur Seine, France). For each video-monitored
experiment we observed the behaviour of the worms during reference periods
(15°C) and heating periods. These observations were aimed at
characterizing specific behaviour during the heating periods. Compared with
previous behavioural studies on other vent organisms
(Shillito et al., 2001;
Ravaux et al., 2003;
Shillito et al., 2006),
whole-body identification of each specimen was very difficult for P.
grasslei because worms tended to aggregate and enlace themselves
(Fig. 1C). For this reason, we
focused on movements of branchial tentacles (beating,
emergence–retraction movement, etc.) for determining survival.
Electrophoresis and immunodetection of HSP70
Samples of the posterior part of the worm body (heat shocked or not) were
ground up in liquid nitrogen, and the powder was homogenized in 1ml of
extraction buffer [50mmoll–1 Tris HCl, pH 7.4; protease
inhibitor cocktail (Sigma, St Quentin Fallavier, France) 1:3 v/v]. The
homogenates were centrifuged at 10000g for 10min at 4°C,
and the extracted proteins were quantified in the supernatant with a Bio-Rad
protein assay (Bio-Rad, Marnes-la-Coquette, France) using bovine serum albumin
(Sigma) as a standard.
For Western blotting, 20 µg of total protein was diluted in loading
buffer [0.1% Tris HCL 0.5 mol l–1 pH 6.8, 0.1% glycerol, 0.2%
SDS (10%), 0.05% β-mercaptoethanol, Bromophenol Blue 0.001%] and
separarated by minigel SDS-PAGE (10% acrylamide:0.3% bisacrylamide w/v). The
proteins were transferred from the SDS-PAGE gel onto a nitrocellulose membrane
using a Mini Trans-Blot Cell (Bio-Rad; 300mA for 45min) using transfer buffer
(25mmoll–1 Tris, 192 mmol l–1 glycine, 20%
isopropanol, pH 8.3).
Dot blot assays were performed in a 48-well plate format using a Bio-Dot microfiltration apparatus (Bio-Rad). Typically, 30 µg of total protein from crude extract were suspended in 200 µl of Tris-buffered saline pH 7.4 (TBS; 50 mmol l–1 NaCl, 150 mmol l–1 Tris) and absorbed onto nitrocellulose membrane by gravity flow. The Bio-Dot was then washed with 200 µl of TBS per well, applying a constant vacuum flow.
Nitrocellulose membranes from the Dot blot and Western blot assays were
then treated similarly for HSP70 detection, following a protocol described
previously (Ravaux et al.,
2003). Density profiles were obtained using a customized plug-in
based on ImageJ software (Abramoff et al.,
2004) that performed an automated background subtraction on the
membrane's image before computing the integrated density of each band.
cDNA amplification and rapid amplification of the 3' and 5' cDNA ends (RACE), and cloning and sequencing of hsp70
Total RNA was extracted from the posterior part of the worm body, and
reverse transcribed to cDNA as previously described
(Ravaux et al., 2007).
The hsp70 sequences were amplified by PCR amplification, using the
primers HSP1, HSP2, HSP3 and HSP4 (Ravaux
et al., 2007) or the primers 5'Primer and 3'Primer,
designed from multiple alignments of homologous sequences (see
Table 2 for primer sequences).
The PCR amplifications were performed following a previously published
protocol (Ravaux et al.,
2007). The 5' and 3' ends of hsp70 cDNA were
obtained using a SMART RACE cDNA amplification kit (Clontech, Mountain View,
CA, USA) with univP and nested primers and the specific primers Pag5, Pag6,
Pag9 and Pag10 for P. grasslei hsp70 form 1, and Heb1 and Heb3 for
Hesiolyra bergi hsp70 (see Table
2).
|
The PCR products were purified using a Geneclean kit (Q-Biogene,
Illkirch-Grassenstaden, France), and subcloned into the pBluescript KS plasmid
vector. The recombined vector was integrated into competent DH5
bacterial cells. Positive colonies were identified by white/blue selection,
and the clones were further screened through PstI/HindIII
(Fermentas, Saint-Rémy-lès-Chevreuse, France) digestion of
plasmid DNA. The sequencing was carried out by Genome Express (Meylan,
France).
Sequence analyses
The sequences were analysed with the TGICL program
(http://compbio.dfci.harvard.edu/tgi/software/)
to find overlapping regions, and assembled into contigs. These sequences were
further analysed using the Expasy Proteomics Server tools
(http://www.expasy.org/).
The nucleotide sequences of the cDNA encoding Paralvinella grasslei hsp70 form 1 and hsp70 form 2 were deposited in GenBank under the accession numbers EF580992 and EF580993, respectively. We also deposited the sequence for Hesiolyra bergi hsp70 under the accession number EF580994. The cDNA sequence obtained for Alvinella pompejana was partial and thus not deposited.
A molecular comparison was carried out between these sequences and those of other annelids: Platynereis dumerii (accession number ABB29585) and Hirudo sp. Since Hirudo sequences are not yet publicly available, we screened a molluscan HSP70 sequence (Mytilus galloprovincialis, AAW52766) against the Hirudo EST database (with the kind permission of Professor M. Salzet, ESA CNRS 8017, Université des Sciences et Technologies de Lille). The 10 best hits were then assembled using TGICL software, yielding one contig, which we used as the Hirudo HSP70 sequence in this paper.
This sequence dataset was automatically aligned by ClustalW and ambiguously aligned regions were removed, leading to 228 amino acid positions. Phylogenetic relationships were then reconstructed by maximum likelihood methods [PHYML (Guindon et al., 2003)] with the following model: JTT substitution matrix, estimated proportion of invariable sites and gamma-distributed substitution rates. The robustness of the topology was assessed by 100 bootstrap replicates in PHYML. Other reconstruction methods, such as parsimony or neighbour joining with the same evolutionary model, produced the same topology (data not shown).
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Table 1 summarizes the survival determined at the end of each video-monitored experiment. At 26 MPa and 15°C, almost all animals were alive after 6 h or 9 h (Expts 2 and 3, respectively; mean survival ± s.d., 91.5±12%). Visual observations throughout these experiments showed a relatively low activity of the worms at 15°C. Most of the time, they were enlaced at the bottom of the cage, agitating their branchial tentacles slowly. Occasionally, they would crawl at the bottom of the cage.
|
Heat shock experiments
Survival and behavioural response
Most of the Paralvinella grasslei survived a 30 min heat shock at
30°C (Expts 4a, 5a and 6a; mean survival ± s.d., 85±15%;
Table 1). Whilst the worms'
activity was low at 15°C, a significant increase in activity was observed
with increasing temperature, and throughout the heat shock. Moreover, some
particular behaviours were detected at 30°C, e.g. worms were seen crawling
actively around the cage (Fig.
1C), and in some cases the worms lifted the anterior part of their
bodies above the substratum. In addition, we observed some P.
grasslei quickly retracting their tentacles and then unfolding them
progressively. This emergence–retraction movement of the worms'
tentacles was very rarely observed at 15°C. More generally, with
increasing temperature, animals tended to move towards the cage sidewalls.
This crawling response was followed by a decrease in activity during the
cooling process, with a progressive return to the reference behaviour.
|
70–75kDa) were detected
with both antibodies. In addition, one high molecular mass band (150kDa),
with a constant low density, was also observed with the polyclonal antibody.
These results confirmed that the chosen antibodies detect HSP70 proteins in
P. grasslei. Dot blot detection and comparison of HSP70 signal intensity, using the polyclonal (Fig. 3B,C) and the monoclonal (data not shown) antibodies, were carried out on a total of 47 tested specimens. The relative difference in the level of HSP70 proteins between groups of R and HS specimens of P. grasslei in response to a 30°C heat shock followed by 0 h (Expts 4a and 4b), 1.5 h (Expts 5a and 5b) and 3.5 h (Expts 6a and 6b) of recovery at 15°C was quantified by densitometry analysis using a plug-in based on ImageJ software (Fig. 3C). HSP70 proteins were detected in all samples and, in spite of important variability between specimens, a significant increase in HSP70 expression following the heat shock was observed. This increase was detected, only when using the polyclonal antibody, in HS individuals that were maintained for 3.5 h at 15°C after the shock (Fig. 3B,C; Mann–Whitney test, U=7, P=0.007). The specimens recovered immediately or 1.5 h after the shock showed similar HSP70 levels in R and HS treatments either with the polyclonal (Fig. 3B,C) or with the monoclonal antibody (data not shown).
Annelid hsp70 sequence analyses
To continue our investigations of the response to heat stress, we
identified cDNAs for hsp70 in P. grasslei. We also isolated
and sequenced the cDNA for hsp70 in the closely related vent annelids
Hesiolyra bergi (full-length cDNA) and Alvinella pompejana
(partial cDNA sequence). A comparison of vent annelid sequences was carried
out with other annelid hsp70 sequences (Platynereis dumerii
accession number ABB29585 and Hirudo sp. assembled from
Hirudo EST database, provided by Professor M. Salzet).
cDNA sequences for vent annelid hsp70
Two full-length cDNAs were obtained from Paralvinella grasslei,
which were arbitrarily named form 1 and form 2.
The cDNA of P. grasslei hsp70 form 1 (GenBank accession number EF580992) is 2207 bp in length, including a 1947 bp coding region and a 3' UTR of 260 bp with a polyadenylation signal sequence (AATAAA at position 2162). The G–C content of the 1947 bp ORF is 47.8%. This ORF encodes a 648 amino acid protein with a predicted molecular mass of 71.65 kDa and a theoretical isoelectric point of 5.74.
The cDNA sequence of P. grasslei hsp70 form 2 (GenBank accession number EF580993) is 2234 bp in length. A single reading frame of 1961 bp is followed by a 273 bp-long 3' UTR, which contains the consensus polyadenylation signal AATAAA located at position 2192. The G–C content of the 1961 bp ORF is 49.4%. This ORF encodes a 653 amino acid protein (Fig. 4) with a calculated molecular mass of 71.38 kDa and a theoretical isoelectric point of 5.23. The cDNA we sequenced from H. bergi belongs to the HSP70 family. This cDNA is 2330 bp in length, including a 3' UTR of 359 bp with the polyadenylation signal sequence at position 2286. The 1971 bp ORF encodes a polypeptide of 656 amino acids with a predicted molecular mass of 71.64 kDa and a theoretical isoelectric point of 5.16. This sequence was deposited in GenBank under the accession number EF580994.
|
A partial cDNA sequence for hsp70 was obtained from Alvinella pompejana using the degenerate primers HSP1, HSP2, HSP3 and HSP4. It is 762 nucleotides in length, which corresponds to a 253 amino acid peptide.
Comparison with other annelid HSP70 proteins
Several general eukaryotic HSP70 family motifs were identified in the amino
acid sequences (see alignment in Fig.
4): three signatures [IDLGTTYS, amino acids 11–18;
IFDLGGGTFDVS(I/V)L, amino acids 199–212; (V/I)VLVGGSTRIPK(I/V)Q, amino
acids 337–350], and a putative ATP–GTP binding site (AEAYLG; amino
acids 133–140). Two additional specific motifs were found: the
non-organellar consensus motif (RARFEEL; amino acids 302–308) and the
cytoplasmic HSP70 carboxyterminal region (GPT(I/V)EEVD).
The two sequences from P. grasslei were clearly different, as confirmed by their position in the tree presented in Fig. 5. This tree was built by a comparison of P. grasslei hsp70 sequences with homologous annelid sequences, and did not aim to reconstruct the hsp70 phylogeny amongst annelids. Indeed, the dataset available for annelid hsp70 is weak, leading to low bootstrap values, except for one node that is highly supported (bootstrap percentage, 100) which corresponds to the group including P. grasslei form 1 and A. pompejana sequences. These two sequences are thus closely related whereas P. grasslei form 2 sequence unambiguously corresponds to a different form of HSP70 protein.
|
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
;
Chevaldonné and Jollivet,
1993; Gaill and Hunt,
1991) (reviewed in Lutz and
Kennish, 1993). This temperature is assumed to be tolerated by the
animals since it is significantly lower than the maxima suggested to be
encountered in the P. grasslei habitat, i.e. up to 40°C in
Alvinella pompejana colonies (Le
Bris et al., 2005).
The almost immediate activity of the worms after repressurization and the high survival rate (91.5±12%) observed for individuals maintained at 15°C at in situ pressure for up to 9 h indicate a relatively good physiological state of the animals.
The oxygen consumption results reflect a metabolic rate of rather active
animals for four P. grasslei out of the five specimens tested
(Fig. 2). Only one worm showed
a size-specific low oxygen uptake level, probably reflecting a trauma induced
by the collection process. The oxygen uptake rates of the four P.
grasslei specimens
(654–1076µlO2g–1DMh–1)
are very similar to those obtained for another deep-sea vent polychaete,
Hesiolyra bergi
[629–1133µlO2g–1DM h–1
(Shillito et al., 2001)] under
the same experimental conditions. When compared with coastal annelids, the
mean oxygen consumption rate for P. grasslei
(787µlO2g–1DMh–1) is higher
than the mean rate obtained at the same temperature for Arenicola
marina [394µlO2g–1DMh–1;
recalculated from the data of Toulmond
(Toulmond, 1975)] or
Nereis diversicolor
[380µlO2g–1DMh–1 (Ivlena
and Popenkina, 1968)], and similar to that of Nephtys hombergi during
its maximal swimming activity
[1000µlO2g–1DMh–1
(Newell and Norcroft, 1967)].
Since P. grasslei did not move actively at 15°C, our results
would be rather high, possibly reflecting stress-induced hyperventilation, or
caused by high oxygen levels in our experiments (surface seawater with an
O2 concentration of ca. 250µmoll–1
compared with the usual O2 concentration in Pacific deep-sea water
of ca. 130µmoll–1
(Johnson et al., 1986;
Johnson et al., 1988;
Millero, 1996) or even lower
near deep-sea vents [ca. 0–100µmoll–1
(Desbruyères et al.,
1998; McCollom,
2000)].
Comparisons of the experimental behaviour of the worms with their natural
behaviour should be made cautiously, since both environment and observation
conditions differ radically. For example, the space available in the cage is
limited and devoid of refuges, in comparison with their natural environment
where a lot of anfractuosities are accessible on the chimney surface.
Moreover, the density of Paralvinella grasslei in the cages
(equivalent to 1300–3300 individuals m–1) is higher
than the density described in situ on the chimney wall (200–800
individuals m–1)
(Chevaldonné and Jollivet,
1993). Another example is the temperature, which is constant in
our experiments whereas it sharply fluctuates in situ
(Le Bris et al., 2005).
Nevertheless, the various types of behaviour observed experimentally resemble
those occurring in their natural habitat. Most of the time, the worms are not
very active, staying inside anfractuosities of the chimney surface and moving
their branchial tentacles slowly. P. grasslei specimens can also move
on the surface of the chimney and are often seen around Alvinella
pompejana tubes (Chevaldonné et
al., 2000).
Considered together, our data suggest that the metabolic rate of P. grasslei is, if not normal, at least far from reflecting that of moribund animals under the conditions of our reference experiments.
Temperature resistance and behavioural response to heat
Paralvinella grasslei can survive up to a 30 min heat shock at
30°C, as demonstrated by the high survival rate determined at the end of
the three heating experiments (85±15%,
Table 1). The survival rate of
heat-shocked animals is comparable to that of reference animals. This
observation shows that the critical thermal maximum (CTmax) of
P. grasslei, defined as the temperature at which the worm is no
longer capable of proper locomotion
(Wehner et al., 1992; Ghering
and Wehner, 1995; Cuculescu et al.,
1998), is above this temperature.
Even if the 30°C shocks were not lethal for P. grasslei, signs
of heat stress were clearly identified. The rise of temperature was
accompanied by an increase in activity of the worms, with frequent
displacements and a greater amplitude of beating of the branchial tentacles.
The emergence–retraction movement of the branchial tentacles, which was
very rare at 15°C, was frequently observed during periods of increasing
temperature. This behaviour has also been described in situ
(Chevaldonné et al., 1993) and Chevaldonné and colleagues
proposed that alvinellids use it for thermoregulation
(Chevaldonné et al.,
1991). At 30°C, a peak of activity was observed and specific
behaviours clearly appeared. The worms were seen actively crawling around the
cage and sometimes lifting their bodies above the substratum. A similar
behaviour was previously described for Hesiolyra bergi, which
actively crawled and swam when the temperature reach 33.5°C, i.e. only a
few degrees before reaching its CTmax at 38°C
(Shillito et al., 2001). In
view of the low activity at 15°C, such behaviours can be inferred as an
escape response to avoid heat zones, and therefore suggest thermal discomfort
of the worms at around 30°C. When compared with other
Paralvinella species living in a similar habitat, this is quite
consistent with the observations on Paralvinella palmiformis, which
avoids temperatures above 35°C, but rather lower than for Paralvinella
sulfincola, which seems to be unaffected by temperatures in the
40–50°C range (Girguis and Lee,
2006). P. grasslei would thus avoid areas at temperatures
above 30°C, where it might be exposed to excessive thermal stress.
Stress protein response
We detected HSP70 proteins in both reference and heat-shocked animals in
all of our experiments (Fig.
3). The signal detected in reference animals was not significantly
different between experiments and may correspond to the presence of the
constitutive form since our antibodies are able to detect both forms.
Alternatively, it may also reflect a `background' response to experimental
stress, like pressure variation upon recovery and conditioning in IPOCAMP,
since HSP70 expression can be triggered by many non-thermal stresses
(Feder and Hofmann, 1999),
including pressure variations (Welch et
al., 1993; Kaarniranta et al.,
2000; Elo et al.,
2005). A great interindividual variability was observed for the
HSP70 signal in each experiment, and especially for heat-shocked animals,
which may reveal a difference in sensitivity to heat between specimens of
P. grasslei (Fig. 3C).
In spite of this variability, a significant increase of HSP70 level was
detected in the heat-shocked specimens maintained for 3.5 h at 15°C after
the shock, when compared with reference animals (see
Fig. 3B). This increase was
detected only when using the polyclonal antibody, which may be explained by
the fact that the polyclonal antibody seems to detect more HSP70 isoforms than
the monoclonal antibody (see preliminary Western blot,
Fig. 3A).
A 30 min heat exposure at 30°C may thus be sufficient to trigger a heat
shock response in P. grasslei. This suggests that the HSP70 enhanced
synthesis threshold in P. grasslei may be lower than 30°C. This
threshold would be in the same range as for the hydrothermal shrimp
Rimicaris exoculata [25°C
(Ravaux et al., 2003)] or the
13°C-acclimated marine snail Tegula funebralis [27°C
(Tomanek and Somero, 1999)].
This temperature would appear to be quite low in view of the temperature that
P. grasslei is supposed to experience in its habitat [up to 60°C
among Alvinella pompejana tubes
(Desbruyères et al.,
1985; Chevaldonné et
al., 1992; Le Bris et al.,
2003; Le Bris et al.,
2005)]. However, in such a highly fluctuating environment, the
relevance of a maximum temperature obtained from a discrete measurement should
still be considered cautiously.
This study is the first attempt to characterize the kinetics of the stress
response in a vent animal. This is nevertheless not a classical kinetic study
because, in order to avoid decompression events when opening the aquarium for
the withdrawal of individuals, several independent experiments were performed
to follow the heat shock response for various times of recovery. Our results
showed a significant increase in the expression of HSP70 occurring 3.5 h after
the heat shock. For comparison, the synthesis of HSP70 in the marine
intertidal snail Tegula brunnea is induced from 2 to 14 h after a
30°C shock, and from 1 to 3 h in its congener Tegula funebralis,
whose body temperature frequently exceeds 30°C during emersion
(Tomanek and Somero, 2000).
Although the heat exposure was shorter for P. grasslei (30 min
versus 2.5 h for the marine snails), the kinetics of the response may
be comparable to that observed for the intertidal species. In view of the
frequent sharp spikes that the worms can encounter in their natural
environment [up to 40°C within a few minutes
(Cary et al., 1998;
Di Meo-Savoie et al., 2004)],
it is surprising to detect so late a heat shock response. However, further
studies are required to determine the time at which the level of HSP70 returns
to a reference level.
Relationship amongst annelid HSP70 proteins
Two hsp70 sequences were identified from P. grasslei,
which clearly correspond to two distinct forms of HSP70. When compared with
homologous vent annelid sequences, the P. grasslei hsp70 form 1 is
closely related to the Alvinella pompejana sequence, whereas the
P. grasslei hsp70 form 2 is rather closer to the Hesiolyra
bergi sequence. Several structural characteristics were proposed to
differentiate hsc70 and hsp70, like the presence/absence of
introns, respectively (Gunther and Walter,
1994; Boutet et al.,
2003; Liu et al.,
2004), or the occurrence of the motif GGMP in the C-terminal
region for HSC70 (Prapapanich et al.,
1996; Liu et al.,
2004). However, according to Leignel and colleagues, since these
criteria are specific for each organism, gene expression studies still seem to
be the only way to distinguish hsp70 and hsc70
(Leignel et al., 2007).
Futures studies on P. grasslei thermal biology should aim to
determine the regulation of expression of these two HSP70 proteins
(constitutive versus inducible) in response to heat shock.
Conclusion
The heat shock experiments performed on P. grasslei suggest that
the CTmax of this species is above 30°C. However, the
significant increase in both the worms' activity and the HSP70 level upon a
30°C exposure gives evidence of thermal stress at this temperature. P.
grasslei specimens may not encounter such severe heat shock in their
natural habitat, but more probably brief spikes at temperatures above
30°C. Since P. grasslei would not exhibit a high tolerance to
elevated temperature, the behavioural response, like escape or
thermoregulatory movements (emergence–retraction of the branchial
tentacles), may be sufficient to prevent exposure to deleterious
temperatures.
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Abramoff, M. D., Magelhaes, P. J. and Ram, S. J. (2004). Image processing with Image J. Biophotonics International 11,36 -42.
Aminot, A. and Chaussepied, M. (1983). Manuel des Analyses Chimiques en Milieu Marin. Brest; CNEXO (Centre National pour l'Exploitation des Océans).
Boutet, I., Tanguy, A. and Morage, D. (2003). Organization and nucleotide sequence of the European flat oyster Ostrea edulis heat shock cognate 70 (hsc70) and heat shock protein (hsp70) genes. Aquat. Toxicol. 65,221 -225.[CrossRef][Medline]
Cary, S. C., Shank, T. and Stein, J. (1998). Worms bask in extreme temperatures. Nature 391,545 -546.[CrossRef]
Chevaldonné, P. and Jollivet, D. (1993). Videoscopic study of deep-sea hydrothermal vent alvinellid polychaete populations: biomass estimation and behaviour. Mar. Ecol. Prog. Ser. 95,251 -262.[CrossRef]
Chevaldonné, P., Desbruyères, D. and Le Haître, M. (1991). Time-series of temperature from three deep-sea hydrothermal vent sites. Deep-Sea Res. 38,1417 -1430.
Chevaldonné, P., Desbruyères, D. and Childress, J. J. (1992). Some like it hot... and some even hotter. Nature 359,593 -594.
Chevaldonné, P., Fisher, C. R., Childress, J. J., Desbruyères, D., Jollivet, D., Zal, F. and Toulmond, A. (2000). Thermotolerance and the `Pompeii worms'. Mar. Ecol. Prog. Ser. 208,293 -295.[CrossRef]
Cuculescu, M., Hyde, D. and Bowler, K. (1998). Thermal tolerance of two species of marine crab, Cancer pagurus et Carcinus maenas. J. Therm. Biol. 23,107 -110.[CrossRef]
Desbruyères, D. and Laubier, L. (1982). Paralvinella grasslei, new genus, new species of alvinellinae (Polychaeta: Ampharetidae) from the Galapagos rift geothermal vents. Proceedings of the Entomological Society of Washington 95,484 -494.
Desbruyères, D., Gaill, F., Laubier, L. and Fouquet, Y. (1985). Polychaetous annelids from hydrothermal vent ecosystems: an ecological overview. Biol. Soc. Washington Bull. 6,103 -116.
Desbruyères, D., Chevaldonné, P., Alayse-Danet, A.-M., Caprais, J.-C., Cosson, R., Gaill, F., Guezennec, J., Hourdez, S., Jollivet, D., Jouin-Toulmond, C. et al. (1998). Burning subjects: biology and ecology of the ``Pompei worm" (Alvinella pompejana Desbruyères et Laubier), a normal dweller of an extreme deep-sea environment. Deep-Sea Res. II 45,383 -422.[CrossRef]
Desbruyères, D., Segonzac, M. and Bright, M. (2006). Handbook of deep-sea hydrothermal vent fauna. IFREMER (Institut Français de la recherche en Mer), Brest.
Di Meo-Savoie, C. A., Luther, G. W. and Cary, S. C. (2004). Physico-chemical characterization of the microhabitat of the epibionts associated with Alvinella pompejana, a hydrothermal vent annelid. Geochim. Cosmochim. Acta 68,2055 -2066.[CrossRef]
Elo, M. A., Kaarniranta, K., Helminen, H. J. and Lammi, M. J. (2005). Hsp90 inhibitor geldanamycin increases hsp70 mRNA stabilisation but fails to activate HSF1 in cells exposed to hydrostatic pressure. Biochimica et Biophysica acta 1743,115 -119.[Medline]
Feder, M. E. and Hofmann, G. E. (1999). Heat shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61,243 -282.[CrossRef][Medline]
Gaill, F. (1993). Aspects of life development at deep-sea hydrothermal vents. FASEB J. 7, 558-565.[Abstract]
Gaill, F. and Hunt, S. (1991). The biology of annelid worms from high temperature hydrothermal vent regions. Rev. Aquat. Sci. 4,107 -137.
Gaill, F., Mann, K., Wiedmann, H., Engel, J. and Timpl, R. (1995). Structural comparison of cuticle and interstitial collagens from annelids living in shallow sea-water and at deep-sea hydrothermal vents. J. Mol. Biol. 246,284 -294.[CrossRef][Medline]
Gehring, W. J. and Wehner, R. (1995). Heat
shock proteins synthesis and thermotolerance in Cataglyphis, an ant from the
sahara desert. Proc. Natl. Acad. Sci. USA
92,2994
-2998.
Girguis, P. R. and Lee, R. (2006). Thermal preference and tolerance of Alvinellids. Nature 312, 231.
Guindon, S. and Gascuel, O. (2003). A simple,
fast, and accurate algorithm to estimate large phylogenies by maximum
likelihood. Syst. Biol.
52,696
-704.
Gunther, E. and Walter, L. (1994). Genetics aspect of the hsp70 multigene family in vertebrates. Experiencia 50,987 -1001.[CrossRef]
Ivleva, I. V. and Popenkina, M. I. (1968). On the temperature dependence of metabolism in poikilothermic animals. In Physiological Principles of the Marine Animals (R. M. Howland, trans.). Marine Biology, ser. no.15, pp.29 -51. Ukraine, Kiev: Biol. Inst. Acad.
Johnson, K. S., Beelher, C. L., Sakamoto-Arnold, C. M. and
Childress, J. J. (1986). In situ measurements of
chemical distributions in a deep sea hydrothermal vent field.
Science 231,1139
-1141.
Johnson, K. S., Childress, J. J. and Beelher, C. L. (1988). Short-term temperature variability in the rose garden hydrothermal vent field: an unstable deep-sea environment. Deep Sea Res. 35,1711 -1727.[CrossRef]
Juniper, K. S., Jonasson, I. R., Tunicliffe, V. and Southward,
A. J. (1992). Influence of tube building polychaete on
hydrothermal chimney mineralization. Geology
20,895
-898.
Kaarniranta, K., Holmberg, C. I., Helminen, H., Eriksson, J., Sistonen, L. and Lammi, M. (2000). Protein synthesis is required for stabilization of hsp70 mRNA upon exposure to both hydrostatic pressurization and elevated temperature. FEBS letters 475,283 -286.[CrossRef][Medline]
Le Bris, N., Sarradin, P.-M. and Caprais, J.-C. (2003). Contrasted sulphide chemistries in the environment of 13°N EPR vent fauna. Deep-Sea Res. Part I 50,737 -747.[CrossRef]
Le Bris, N., Zbinden, M. and Gaill, F. (2005). Processes controlling the physico-chemical micro-environments associated with Pompeii worms. Deep Sea Res. Part I 52,1071 -1083.[CrossRef]
Leignel, V., Cibois, M., Moreau, B. and Chénais, B. (2007). Identification of new subgroup of HSP70 in Bythograeidae (hydrothermal crabs) and Xanthidae. Gene 396, 84-92.[CrossRef][Medline]
Liu, J., Yang, W. J., Zhu, X. J., Karouna-Renier, N. K. and Rao, R. K. (2004). Molecular cloning and expression of two HSP70 genes in the prawn, Macrobrachium rosenbergii. Cell Stress Chaperones 9,313 -323.[CrossRef][Medline]
Lutz, R. A. and Kennish, M. J. (1993). Ecology of deep-sea hydrothermal vent communities: a review. Rev. Geophys. 31,211 -242.[CrossRef]
McCollom, T. M. (2000). Geochemical constraints on primary productivity in submarine hydrothermal vent plumes. Deep Sea Res. Part I 47,85 -101.[CrossRef]
Millero, F. J. (1996). Marine Science Series: Chemical Oceanography (ed. M. J. Kennish and R. A. Lutz), pp.219 -229. Boca Raton, FL; CRC Press.
Newell, R. C. and Norcroft, H. C. (1967). A re-interpretation of the effect of temperature on the metabolism of certain marine invertebrates. J. Zool. 151,277 -298.
Parsell, D. A. and Lindquist, S. (1993). The function of heat shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27,437 -496.[CrossRef][Medline]
Prapapanich, V., Chen, S., Toran, E. J., Rimerman, R. A. and Smith, D. F. (1996). Mutational analysis of the hsp70-interacting protein Hip. Mol. Cell. Biol. 16,6200 -6207.[Abstract]
Ravaux, J., Gaill, F., Le Bris, N., Sarradin, P.-M., Jollivet,
D. and Shillito, B. (2003). Heat-Shock response and
temperature resistance in the deep-sea vent shrimp Rimicaris exoculata.J. Exp. Biol. 206,2345
-2354.
Ravaux, J., Toullec, J.-Y., Léger, N., Lopez, P., Gaill, F. and Shillito, B. (2007). First hsp70 from hydrothermal vent shrimps, Mirocaris fortunata and Rimicaris exoculata: characterization and sequence analysis. Gene 386,162 -172.[CrossRef][Medline]
Sarradin, P.-M., Caprais, J.-C., Briand, P., Gaill, F., Shillito, B. and Desbruyères, D. (1998). Chemical and thermal description of the environment of the Genesis hydrothermal vent community (13°N, EPR). Cah. Biol. Mar. 39,159 -167.
Shillito, B., Jollivet, D., Sarradin, P.-M., Rodier, P., Lallier, F., Desbruyères, D. and Gaill, F. (2001). Temperature resistance of Hesiolyra bergi, a polychaetous annelid living on deap-sea vent smoker walls. Mar. Ecol. Prog. Ser. 216,141 -149.[CrossRef]
Shillito, B., Le Bris, N., Gaill, F., Rees, J.-F. and Zal, F. (2004). First access to live Alvinellas. High Pres. Res. 24,169 -172.[CrossRef]
Shillito, B., Le Bris, N., Hourdez, S., Ravaux, J., Cottin, D.,
Caprais, J.-C., Jollivet, D. and Gaill, F. (2006).
Temperature resistance studies on the deep-sea vent shrimp Mirocaris
fortunata. J. Exp. Biol.
209,945
-955.
Tomanek, L. and Somero, G. N. (1999). Evolutionary and acclimation-induced variation in the heat shock responses of congeneric marine snails (Genus Tegula) from different thermal habitats: implications for limits of thermotolerance and biogeography. J. Exp. Biol. 202,2925 -2936.[Abstract]
Tomanek, L. and Somero, G. N. (2000). Time course and magnitude of synthesis of heat shock proteins in congeneric marine snails (genus Tegula) from different tidal heights. Physiol. Biochem. Zool. 73,249 -256.[CrossRef][Medline]
Toulmond, A. (1975). Blood oxygen transport and
metabolism of the confined lugworm Arenicola marina (L.).
J. Exp. Biol. 63,647
-660.
Tunnicliffe, V. (1991). The biology of hydrothermal vents: ecology and evolution. Oceanogr. Mar. Biol. A Rev. 29,319 -407.
Wehner, R., March, A. C. and Wehner, S. (1992). Desert ants on a thermal tightrope. Nature 357,586 -587.[CrossRef]
Welch, T. J., Farewell, A., Neidhardt, F. C. and Bartlett, D.
H. (1993). Stress response of Escherichia coli to
elevated hydrostatic pressure. J. Bacteriol.
175,7170
-7177.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
