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Heat-shock response and temperature resistance in the deep-sea vent shrimp Rimicaris exoculata

Juliette Ravaux¹, Françoise Gaill¹, Nadine Le Bris², Pierre-Marie Sarradin², Didier Jollivet³ and Bruce Shillito^1,*

¹ UMR CNRS 7138 `Systématique, Adaptation et Evolution', Université Pierre et Marie Curie, 7 Quai St-Bernard, Batiment A, 75252 Paris Cedex 05, France
<sup>2</sup> Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Centre de Brest, DRO-EP, bp 70, 29280 Plouzané, France
<sup>3</sup> Station Marine de Roscoff, UPR CNRS 9042, Equipe `Evolution et génétique des populations marines', bp 74, 29682 Roscoff Cedex, France

View larger version (44K): [in a new window]   Fig. 1. Experimental set-up in the IPOCAMP (Incubateur Pressurisé pour l'Observation et la Culture d'Animaux Marins Profonds) pressure vessel for heat-exposure experiments. (A) The pressure vessel contains three cages (only one is represented here). Each cage is a PVC cylinder that is closed at the top with an inclined transparent Plexiglas lid (about 9 cm diameter; 15 cm at the highest point). Ventilation holes are located at the top and bottom of each cage, with Pt-100 probes that are positioned in the water flow, allowing direct reading of the upstream (t1) and downstream (t2) temperatures. Connections in the lid of the pressure vessel are terminated by sapphire windows. By inserting an endoscope into the appropriate connection, the content of one of the cages may be observed. Illumination is achieved through the other connections by means of optical fiber light-guides. Large arrows indicate the inlet and outlet of circulating seawater, which is forced through the cage ventilation holes. The internal diameter of the pressure vessel is 20 cm, while its height is approximately 60 cm. (B) Video-view of a nylon-mesh cage containing five Rimicaris exoculata specimens, maintained in the IPOCAMP at in situ pressure (230x105 Pa) and a temperature of 15°C. The white V-shaped ocular plates (`eyes') of the shrimps are clearly visible for two individuals that are resting on the bottom of the cage. Three other specimens (on the left of the image) are resting on the vertical wall at the top of the cage. Diameter of cage bottom = 9 cm, approximate length of shrimps = 3–6 cm.

View larger version (16K): [in a new window]   Fig. 2. Video analyses of Rimicaris exoculata behavior during in vivo experiments. Behavior during 30 s sequences, as a function of time since re-pressurization: , `motionless'; +, `moving'; , `active walking or swimming'; •, `spasms' (see Materials and methods). (A,B) Behavior (lower graphs) related to temperature (upper graphs, mean temperature between t1 and t2 probes; see Fig. 1A) during two heat-exposure experiments [Expts 3 (A) and 4 (B); 15 individuals per experiment, split into three groups of five individuals]. For each observation time, the maximal error regarding the corresponding temperature is approximately ±2°C (A) and ±1.7°C (B).

View larger version (10K): [in a new window]   Fig. 3. Oxygen uptake in Rimicaris exoculata in relation to shrimp dry mass for seven individuals maintained at in situ pressure (3 h or 4 h; 15°C; 230x105 Pa; Expt 1; see Materials and methods). The oxygen consumption rates (R; expressed in µg O2 h-1) correlate to dry mass (M; expressed in mg dry mass) of individuals following the equation: R=1.748M0.891 (r=0.832, N=7, P<0.05).

View larger version (55K): [in a new window]   Fig. 4. Protein profile of heat-shocked and reference Rimicaris exoculata abdomen samples (4 µg total protein well-1) as shown on a silver-stained 10% gel. The `reference' lane shows individuals from Expt 2 (24 h; 15°C); the `heat-shock' lane shows individuals from Expt 4 (24 h; 25°C heat shock; see Materials and methods). Arrowheads indicate bands of interest that are not apparent or are poorly represented in the reference samples and are visible in the heat-shocked samples at molecular masses of approximately 205 kDa, 90 kDa and 70 kDa.

View larger version (23K): [in a new window]   Fig. 5. (A) Western blot of Rimicaris exoculata abdomen samples for the detection of hsp70 proteins (anti-human hsp70). Each lane corresponds to a different individual. The `reference' lanes show individuals from Expt 2 (24 h; 15°C); the `heat-shock' lanes show individuals from Expt 4 (24 h; 25°C heat shock; see Materials and methods). Arrowheads indicate the hsp70 proteins, named band HMM (high molecular mass) and LMM (low molecular mass). (B) Comparison of hsp70 levels in abdomen samples as estimated by density measurements of the western blot protein bands (HMM and LMM). R = reference samples, HS = heat-shocked samples. Density profiles were obtained from the western blot membranes using NIH Image 1.6 software. The density of each band (expressed in arbitrary units) was calculated from the area of the corresponding peak on the profile. Each column represents the mean of band density for four individuals (± S.D.). The asterisk indicates a significant difference between treatments (Mann–Whitney test, U=0.0, P=0.021).