Here is a complete(?) summary of the experiment
Oysters
235 adults: from Pacific Hybreed, 40-100mm
- 4 families
- 30 diploid
- 30 triploid
a few missing, hence n≠240. These diploids and triploids are siblings. These groups of 30 were given a color or animal name.
120 juveniles: USDA 2023 spawn, 20-55mm
120 seed: USDA 2023 spawn, 6-15mm
roughly 120 spat: USDA 2023 spawn, 1-6mm
Experiment
Half from each group were put into a treatment tank, half into a control tank.
| control: | treatment: |
|---|---|
| 15 fam1 diploid | 15 fam1 diploid |
| 15 fam1 triploid | 15 fam1 triploid |
| 15 fam2 diploid | 15 fam2 diploid |
| 15 fam2 triploid | 15 fam2 triploid |
| 15 fam3 diploid | 15 fam3 diploid |
| 15 fam3 triploid | 15 fam3 triploid |
| 15 fam4 diploid | 15 fam4 diploid |
| 15 fam4 triploid | 15 fam4 triploid |
| 60 juveniles (usda, mixed families) | 60 juveniles (usda, mixed families) |
| 60 seed (usda, mixed families) | 60 seed (usda, mixed families) |
| ~60 spat (usda, mixed families) | ~60 spat (usda, mixed families) |
Adults kept in bags at the bottom of the tanks. Seed and spat were kept in tubes, and juveniles were kept in tubes within tubes, they won’t be in water that has been exposed to other groups.
treatment left, control right.
The control tank was left at ambient temp, around 17ºC. The treatment tank was heated (+2ºC per hour) up to 26ºC, for 6 hours a day, for 7 weeks (began 10 2 2023, ended 11 21 2023). Tubes were cleaned semi-regularly
Adults and juveniles mechanically stressed via salad spinner for 15 minutes on these dates: 10/2/2023, 10/4/2023, 10/11/2023
Lengths recorded periodically.
On 11/21/2023:
4 groups (dog, yellow, cat, toad) (n=120) were sampled for later RNA and DNA analysis.
We took 7 from a group of 15 (e.g 7 from “fam1 diploid” in “control”) and immediately sampled those. We cut gill tissue and mantle tissue, placed in separate tubes each with RNA later. We also put tissue into a tube with 70% etOH for DNA analysis. Efforts were taken to minimize cross-contamination (clean gloves and tools).
The other 8 from this group of 15 were placed into a bucket of seawater at 32ºC for 30 minutes, then tumbled for 5 minutes in a salad spinner. They were immediately sampled in the same fashion.
We repeated this for the equivalent in each tank (e.g if we did this for we also did this for fam1 diploid in control, we also did this for fam1 diploid in treatment).
Heaters were turned off and treatment tank was left at ambient.
On 2/28/2024:
We repeated this process from 11/21/2023 but with some differences. Namely:
- Sampled all seed, all spat, and all juveniles
- Did not sample remaining Adults
- Only gill tissue was taken
- No mechanical stress
- tubes for seed and spat are individuals, no multiple samples from the same oysters. They were too small.
- flash froze additional samples for potential proteomic/metabolomic analysis