Materials and Methods
- Proteomic Analysis of Mussels Exposed to Fresh and Weathered Prestige’s Oil

Animal Collection and Experimental Procedure Mussels, M. galloprovincialis, 3.5 to 4.5 cm shell length and of undetermined sex were collected at low tide from ten different sampling sites in the NW and NE of the Iberian Peninsula in July 2005 for the field studies, and in a NE location in September 2005 for the laboratory experiments. Sampling sites in the NW were Sao Bartolomeu do Mar (41°34’36’’N, 8°48’2’’W) (from now on referred as Sao Bartolomeu), Aguiño (42°31’13’’N, 9°0’36’’W), Caldebarcos (42°50’48’’N, 9°7’52’’W), Camelle (43°11’38’’N, 9°5’48’’W), and Segaño (43°27’21’’N, 8°18’34’’W). Sampling sites in the NE were Muskiz (43°21’32’’N, 3°6’40’’W), Arrigunaga (43°21’17’’N, 3°1’11’’W), Gorliz (43°25’7’’N, 2°56’51’’W), Mundaka (43°24’16’’N, 2°41’43’’W), and Hondarribia (43°22’40’’N, 1°47’24’’W). Mussels for the laboratory experiment were collected from Mundaka (43°24’16’’N, 2°41’43’’W), a relatively clean location in the mouth of the Biosphere Reserve of Urdaibai estuary (Orbea and Cajaraville, 2006). Sampling sites are summarized in Figure 1.

Mussels collected from Mundaka for the laboratory experiment were acclimatized in the laboratory for 15 days and afterwards divided in three high-density polyethylene tanks at a mussel density of one mussel per three liters of seawater. Water temperature was kept at 20 °C, salinity at 35‰ and oxygen levels above 6 mg/L by constant aeration. A photoperiod of 11 hours was set and commercial food (JBL KorallFluid, JBL BmgH & Co. KG, Neuhofen, Germany) provided every day. The heavy fuel oil that is similar to that spilled by the Prestige (IFO 380, marine fuel RMG 35-ISO 8217) was provided by the Vigo Technical Office Against Accidental Marine Spills (University of Vigo, Spain). Oily sediments were prepared by mixing 150 mL oil with 5 kg gravel, and 6 kg sand, and placed on the bottom of the tanks. Weathered fuel oil (WF) was obtained by letting the sediment stand in a water-filled tank during two and a half months. Fresh fuel oil (FF) was obtained by adding the sediment to a water filled tank precisely before the exposure started. Exposure to FF tried to mimic the situation in the most affected areas in the NW immediately after the Prestige’s oil spill, whereas exposure to WF would mimic the situation in any of the sampling sites months after the spill. Mussels were also kept in a control tank where no oil was added.

For our experiments, four mussels were collected from each sampling site and eight from each tank: four after two days of exposure, and four after 16 days had passed. Digestive glands were immediately dissected out and frozen in liquid nitrogen in situ in all the cases, and kept at - 80 °C until the proteomics analysis.

Proteomics Analysis

Digestive glands were processed following a protocol for sample prefractionation and 2-DE protein separation already described (Amelina et al., 2007). Briefly, digestive glands were homogenized with the aid of a pestle and AG®501- X8 Resin glass beads (BioRad Laboratories, Inc., Hercules, CA, USA) in a homogenization buffer containing a protease inhibitor cocktail. Following homogenization, a three-step centrifugation was applied and an organelle-enriched fraction therefore obtained. Low-abundant proteins were then obtained by an anion-exchange chromatography in batch using Q-sepharose™ Fast Flow (Amersham Biosciences AB, GE Healthcare, Uppsala, Sweden).

Proteins from the eluted fractions were then precipitated by the addition of 20% trichloroacetic acid in 100% cold acetone containing 0.07% β-mercaptoethanol, and the precipitate was washed with 100% cold acetone containing 0.07% β-mercaptoethanol. Precipitated proteins were solubilized in a solubilization buffer described by Rabilloud with some modifications (Rabilloud, 1998; Amelina et al., 2007), alkylated with 30 mM iodoacetamide (IAA) in darkness and mixed with a rehydration buffer previous to the isoelectrofocusing (IEF) step. Proteins (300 µg) were loaded in the PROTEAN® IEF Cell (BioRad Laboratories) tray and IPG strips (11 cm, pH range of 4-7, BioRad Laboratories) placed on top. The following program was followed: passive rehydration for 12 h at 50 V and 20 °C, 250 V for 15 min, rapid voltage ramping to obtain 8,000 V and a final focusing at 8,000 V until 35,000 V.h were achieved. The focusing was held at 500 V until strips were removed from the tray. In all the steps, a maximum current limit of 50 µA per strip was established. IPG strips were first reduced (1% dithiothreitol (w/v)), and then alkylated (4% IAA (w/v)) in an equilibration buffer (Amelina et al., 2007) previous to SDS-PAGE.

Equilibrated IPG strips were laid on top of homogeneous 12.5% Tris-HCl Criterion™ Precast Gels (BioRad Laboratories) and SDS-PAGE run at 120 V. 2- DE gels were fixed and stained with CBB G-250 for 12-18 h. Distained 2-DE gels were scanned in a UMAX Image Scanner (Amersham Biosciences) and analyzed by ImageMaster™ 2D Platinum 6.0 (Amersham Biosciences). 2-DE gel images were cropped, spots automatically detected, wrong detections manually corrected and finally the volume % (vol%) of each spot calculated based on the total spot volume in each gel. A master gel was chosen for each sampling site and exposure group. Spots from the rest of the three gels inside each sampling site/group were then matched to the master gel. Higher-level match-sets were constructed between master gels. Image analyses of the field study and the laboratory exposure were separately performed, but their highest-level master images were finally matched between them.

Statistical Analysis

Vol% data was exported to SAS® 9.1.9 (SAS Institute Inc., Cary, NC, USA) and MATLAB® 7.5.0 (The MathWorks, Inc., Natick, MA, USA) for the statistical analyses. In total, 468 spots were obtained in the match set from the laboratory exposure experiment. Missing values in the data set came from spots with intensities lower than the detection limit of the image analyzer, or from spots absent in the 2-DE gels, but not from an incorrect matching. Therefore, zero values were input. In the few cases where the missing value happened in a group with relatively high values, the mean value of the three replicates from the group was input.

Two-way ANOVA was performed on each spot separately to extract those spots that differed among the groups, based on the following linear effects model:

i = 1,2; j = 1, 2, 3; and k = 1, 2, 3, 4, where α is the time effect average over treatments, β is the treatment effect average over time, γ is the interaction effect, and ε is the variation within each group of 4 replicates, εijk ~ N (0, σij). The response variable y is the value of the specific spot. On account of performing multiple tests, there will necessarily be a number of false positives. By use of the False Discovery Rate (FDR) procedure (Hochberg and Benjamini, 1995) we can protect against too many false positives. FDR was set to 5 %.

PCA is a multivariate statistics technique that takes into account a group of variables instead of focusing in one variable at a time, as is the case for univariate analyses. PCA was used to find out if there was any structure in the data selected after the ANOVA and FRD analyses that could explain differences among the exposure groups. A covariance matrix where each spot was set as a variable and each gel as an observation was used to extract the principal components. In order to improve the PCA outcome, several spots were removed from the dataset.

Finally, the vol% of the selected spots was obtained from the field experiment data. A putative group membership for the different sampling sites was obtained based on the new variable’s proximity to the experimental variables that were separated by the PCA.

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