Sterile syringe

At sacrifice, blood samples were collected to measure the serum concentrations of total cholesterol, TG, very-low-density lipoprotein (VLDL), LDL, high-density lipoprotein (HDL), glucose, and adiponectin after overnight fasting from 10 or 14 mice in each group. Whole blood anti-coagulated with heparin lithium was taken from the inferior vena cava with a sterile syringe (Terumo, Tokyo, Japan). The serum was obtained by centrifugation (3000 rpm for 10 min), and stored at −80 °C until measurement. The serum TG and total cholesterol levels were determined using commercial enzymatic assay kits (TG, L-Type WAKO-TG·H; and total cholesterol, L-Type WAKO-CHO·H), obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The serum levels of HDL, LDL, and VLDL were determined using an HDL and LDL/VLDL Cholesterol Quantitation Kit (BioVision, Inc., Milpitas, CA, USA). The serum glucose level was determined using commercial enzymatic assay kit (Glucose CII-test WAKO, Wako Pure Chemical Industries). These measurements were expressed as mg/dL. The serum adiponectin level (µg/mL) was determined with Mouse/Rat Adiponectin ELISA kits (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan).

Samples were collected with a sterile syringe (TERUMO Corporation), connected to bioreactors by a Norprene tube, for chemical and biological analyses. The sampling system was purged before each sampling. 10 mL samples were collected for chemical analysis from each reactor along the incubation at 5.4 (i.e., 10 h after oil addition), 7, 8, 10, 11, and 15 days of incubation. Sampling was performed before switching condition. Samples were stored in amber glass bottles with polytetrafluoroethylene stoppers (WHEATON) at -20°C. For molecular analyses 1.5 mL of slurry was collected and immediately mixed with 190 μL of RNA stabilization solution (the RNA stabilization buffer was prepared as follow: 5 mL of phenol were mixed with 5 mL of 1 M Na-acetate buffer pH 5.5, then the two phases were separated by centrifugation at 4000 × g for 3 min. The phenolic phase was finally added to 95 mL of pure ethanol) to preserve rRNA transcripts integrity. Samples with RNA stabilization solution were homogenized and centrifuged at 10000 × g for 5 min at 4°C (Jouan MR 1812). The supernatant was then removed and tubes containing pellets were introduced immediately in liquid nitrogen and stored at -80°C. Samples were collected in triplicate from each reactor at days 0, 5 (before oil addition), 5.4 (10 h after oil addition), 7, 8, 10, 11, and 15.

For both families (A1 and A2), haemolymph was collected from six live oysters with a sterile syringe (1 ml, Terumo) from the pericardial cavity and pooled for each tank, condition and family. These samples were collected at 0.5, 10, 26, 72 and 144 hours post-infection (hpi). Haemolymph pools were centrifuged at 300 rpm during 10 min at 4 °C then filtered at 0.22 μm in order to study antiviral activity. All plasma samples (without haemocytes) were stored at −80 °C.
Two pieces of mantle were sampled from two live individuals per tank (6 individuals per time/condition) from both families. A piece of mantle (50 to 100 mg) was disposed in a tube containing 1 mL of TRIZOL® Reagent™ (Ambion®) and frozen at −80 °C for further RNA extraction. The second piece of mantle was directly frozen at −20 °C for DNA extraction.