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
Delphine Cottin1,2,
Juliette Ravaux1,2,*,
Nelly Léger1,2,
Sébastien Halary1,2,
Jean-Yves Toullec3,4,
Pierre-Marie Sarradin5,
Françoise Gaill1,2 and
Bruce Shillito1,2
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
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Fig. 1. Experimental set-up for in vivo experiments at in situ
pressure (26 MPa) (modified from Ravaux et
al., 2003 ). (A) The pressure vessel IPOCAMP (Incubateur
Pressurisé pour l'Observation et la Culture d'Animaux Marins Profonds;
internal diameter 20 cm, height 60 cm) contains two PVC cages (length 6 cm,
width 5 cm, height 6 cm) closed at the top with a transparent polyethylene
lid. Three sapphire windows in the pressure vessel lid allow the insertion of
an endoscope and two optical-fibre light-guides, for the observation of the
inside of the cages. Large arrows indicate the inlet and outlet of circulating
seawater, which is forced through the cage ventilation holes via a
PVC tube. Two Pt100 probes are positioned in the water flow indicating the
upstream (T1) and the downstream (T2) temperature. (B) Temperature profiles
for a typical heat shock (HS) experiment (solid line, T1 probe; broken line,
T2 probe; bold line, mean temperature obtained from T1 and T2 probes). The
animals were maintained at 15°C and subsequently exposed to a sharp heat
shock at 30–33°C. After rapid cooling to 15°C, the animals were
recovered at different times after the heat shock (see Materials and methods
for more details). (C) Video view of a cage containing eight P.
grasslei specimens, maintained at a temperature of ca. 30°C
during a HS experiment. The approximate length of P. grasslei
specimens was 7 cm.
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Fig. 2. Oxygen consumption (R; µlO2h–1) as
a function of dry mass (DM; mg) for five Paralvinella grasslei
maintained at in situ pressure (26 MPa, 15°C, 6 h; Expt 1).
R correlates with DM of individuals following the equation:
R=7.07DM0.37 (r=0.94, N=5,
P<0.01).
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Fig. 3. (A) Western blot profiles obtained for the detection of HSP70 proteins in
P. grasslei using the anti-rat polyclonal antibody (lane 1) or the
anti-chicken monoclonal antibody (lane 2). Several bands with a molecular mass
between 70 and 75 kDa were detected for both antibodies. A weak band around
150 kDa was also observed with the polyclonal antibody. (B) Dot blot detection
of HSP70 proteins in P. grasslei using the anti-rat polyclonal HSP70
antibody. Each band corresponds to a different individual. The first two
columns represent reference (R) and heat-shocked (HS) individuals frozen
immediately after the shock (Expts 4a and 4b; 0 h). The two central columns
show R and HS specimens that were maintained for 1.5 h at 15°C after the
shock (Expts 5a and 5b, 1.5 h). Finally, the two last columns show R and HS
animals maintained for 3.5 h at 15°C after the heat shock (Expts 6a and
6b, 3.5 h). (C) Dot blot signal intensity comparison of HSP70 levels for R
(grey columns) and HS (black columns) P. grasslei. The density of
each band (expressed in arbitrary units, a.u.) was calculated using a plug-in
based on ImageJ software. Each column represents the mean of the band density
(±s.d.) for the eight R or HS individuals from the corresponding column
above (except for Expt 6a where only seven P. grasslei were used; the
dash shows the empty well). The asterisk indicates a significant difference
between treatments (Mann–Whitney test; U=7,
P=0.007).
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Fig. 4. Comparison of the HSP70 sequences used for phylogenetic analyses. 1,
Paralvinella grasslei form 1; 2, Paralvinella grasslei form
2; 3, Hesiolyra bergi; 4, Hirudo sp.; 5, Platynereis
dumerii; 6, Alvinella pompejana; 7, Mytilus
galloprovincialis; 8, Mytilus galloprovincialis. The residues
corresponding to the three HSP70 family signatures are boxed. The residues
used for building the tree are indicated in bold (228 amino acid positions).
The sequences used for rooting the tree are Mytilus galloprovincialis
HSP70 (accession no. AAW52766) and HSC70 (accession no. CAH04109).
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Fig. 5. Relationships amongst annelid HSP70 proteins. The tree was reconstructed by
maximum likelihood methods [PHYML (Guindon et al., 2003)] under a
JTT+invariant+gamma model (see Materials and methods). The accession number of
the amino acid sequences is provided after each species name. The values
indicated on the branches correspond to bootstrap percentages. According to
the tree, the sequence Paralvinella grasslei form 1 is clearly
related to the Alvinella pompejana sequence, while the
Paralvinella grasslei form 2 sequence is unambiguously distinct from
this group.
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© The Company of Biologists Ltd 2008
