---
title: "Preliminary Project Outline"
author: "Kathleen Durkin"
date: "2024-06-19"
categories: ["pacific_oyster"]
format:
  html:
    toc: true
engine: knitr
bibliography: ../../../references.bib
---

### 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:

1.  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.
2.  MHW definition is pretty distinct, would need to decide what type of scenario to simulate
3.  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. 
4.  Apparently heartbeat is potentially a better metric for metabolic rate than respiration, and is nondestructive
5.  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
6.  Should also keep in mind that genetic composition can completely change the true sample size (e.g., if triploids and diploids are siblings)
7.  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)

-   Say manual hardening 3-4 hrs/day MWF. Then 2 wk recovery and MHW of 40C immersed and emmersed for a few hours, then montier at ambient for a few weeks. Can stress test rest if alive.

-   Also, can include a much larger number of diploids and outplant MHW survivors to test long term effects

-   could throw in the tetraploids to spawn at the end and see if larvae are more resilient

-   We need to grab 4 cohorts that havent been tested yet (can see github issue about ploidy stuff)
