Pre-Experiment
Oysters
- 8°C
- fed daily with 5-10 ml per tank commercial shellfish diet (Isochrysis 1800 mix Marine Algae)
- Partial water changes once per week (remove 5 gallons, replace with fresh 5 gallons)
- Seawater from Seattle Aquarium
- Aeration with aquarium aerator/stone
- circulation with underwater pump
- Tanks 10 gallon plastic storage tubs (costco storage tubs)
Homogenization tubes for ctenidia prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, and oyster # (1,2,3...)
Collection 1.5 ml tubes (screw cap) for ctenidia prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, oyster # (1,2,3...), and tissue type (C, M)
Collection 1.5 ml tubes (screw cap) for mantle prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, oyster # (1,2,3...), and tissue type (C, M)
15 ml conical tubes for whole oyster prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, and oyster # (1,2,3...)
Warm fresh seawater in 500 ml beaker to 38°C by immersing it in a water bath at 38°C.
Experiment
Heat Stress
- 22 oysters each pop in a mesh bag were lowered (stagger each pop by 1 hour) into 500 ml of prewarmed 38°C sea water (1 hr timer started)
- N pop first, H pop next, S pop last
- after 1 hour exposure heat stress ends, animals returned to 8°C water.
- 1 hour post exposure 8 animals sampled from each group staggered by 1 hour
- N pop first, H pop next, S pop last
- collected with ethanol flame sterilized forceps and scissors
- tools kept in 100% EtOH
- ran through EtOH flame
- allow EtOH to burn off
- Collect tissue
- replace in 100% EtOH
- wipe occasionally with paper towel and resterilize
- ctenidia sample (50-100 mg) homogenized in 1 ml Trizol stored at -80°C
- 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
- mantle sample (decent size) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
- remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
- shells in individual labeled ziploc bags so correspond to sample including oyster id
Mechanical Stress
- 22 oysters from 1 pop placed in bucket in centrifuge and spun for 5 minutes at 1000 rpm. Samples staggered by 1 hour. Completed after all heat shock treatments and samples were taken.
- N pop first, H pop next, S pop last
- after 5 minutes of mechanical stress, animals returned to 8°C water
- 1 hour post exposure 8 animals sampled from each group staggered by 1 hour
- N pop first, H pop next, S pop last
- collected with ethanol flame sterilized forceps and scissors
- tools kept in 100% EtOH
- ran through EtOH flame
- allow EtOH to burn off
- Collect tissue
- replace in 100% EtOH
- wipe occasionally with paper towel and resterilize
- ctenidia sample (50-100 mg) homogenized in 1 ml Trizol stored at -80°C
- 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
- mantle sample (decent size) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
- remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
- shells in individual labeled ziploc bags so correspond to sample including oyster id
