Say I had 100 diploids and 100 triploids…
Acclimation: Hold in lab ambient conditions for 2 weeks. Measure size and body condition.
Priming: Control (50 2N, 50 3N), expose to 25C 6hr/day for 2 weeks (50 2N, 50 3N)
Sample: Evaluate mortality, size, and body condition of all oysters. Retain 10 samples from each ploidy/treatment combo (40 total). Resazurin assay. Evaluate reproductive stage. Tissue samples to be used for assay(s) of choice (e.g. glycogen content) and, ideally, some reserved for RNAseq.
Recovery: Hold at ambient for 2 weeks
MHW simulation: Ramp up to 30C and hold remaining oysters (40 2N control, 40 3N control; 40 2N hardened, 40 3N hardened) at 30C for 72 hours. At end, note mortality.
If <10 live individuals remaining in each treatment combo: Sample remaining oysters as described above.
If >10 live individuals in all treatment combos: Sample remaining oysters from each group, keeping at least 10 in each combo. Perform survivorship test with n=10 from each combo (2N control, 3N control, 2N hardened, 3N hardened), holding at 40C until mortality and noting time to mortality for each.
Questions/Notes:
- We'd need to make sure they don't spawn, so need to do some digging into what conditions might induce spawning. Would be important during priming stage.
- MHW definition is pretty distinct, would need to decide what type of scenario to simulate
- Need to figure out sample sizes for whatever tests I want to do. Would be benefitted by running some test assays before starting experiment. A bunch of these assays have been used with Pacific oysters already, so could find those and make a table or something.
- Apparently heartbeat is potentially a better metric for metabolic rate than respiration, and is nondestructive
- Acute stress test could be a good biomarker itself. Aka take an oyster after hardening and see how long it survives at 35C, monitoring heartbeat and stuff. Would, of course, also want to do preliminary tests
- Should also keep in mind that genetic composition can completely change the true sample size (e.g., if triploids and diploids are siblings)
- Should look at all the hardening papers to try to assess best timeline (e.g. how long to harden, how long to wait between hardening and stress)