This post details a plan for running acute stress survival assays in the lab the week of December 8-12 from Westcott oysters.

I’ll continue to edit this post as we revise our plan and add data.

Overview

Our goal is to subsample oysters from the treatments at Westcott and conduct acute survival testing in the lab at UW. This post describes the plan for survival testing. We may also try to do some resazurin work on gill tissue, but this will be preliminary.

Our hypothesis is that hardened oysters will have higher survival under thermal stress than control oysters.

General Timeline

Below is a general timeline of events. I have specific information for scheduling below.

• November 26: Ariana put together supplies and conduct resazurin tests.
• December 2: Assessment at Westcott and taking subsamples for transport
• December 3-4: Grace L. transport Westcott oysters to UW
• December 4: Noah O. receives oysters in the lab at UW and makes sure tanks are running and oysters are fed
• December 8: Experiment set up and preparation
• December 9-11: Survival testing
• December 12: Clean up and storage of any remaining samples

People available to help: Steven, Christina, Noah O., Ariana (Dec 10-11 only)

tl;dr: what to do at UW during testing

Monday Dec 8

Today is experiment set up and preparation! Here are the things to do:

• Label plastic cups (drinking sized cups, plastic, ordered by Steven) with a number with permanent marker. Label them with numbers 1-120.
• Make three 5-gallon buckets full of Instant Ocean seawater at salinity psu=20-25. Leave stored in cold room.
• Familiarize yourself with the oysters. There are four bags of oysters from Westcott in the tanks. There are 30 oysters in each bag. Each bag contains a different treatment group. 20 oysters from each bag will go into cups at high temperature and 10 oysters from each bag will go into cups at control temperature. There will be 1 oyster per cup.
• Familiarize yourself with the data sheet. Ariana left a printed datasheet on the counter in FTR 213. For each oyster, you will record the cup number, group information/tag nubmer of the bag, the date trial started, and whether the oyster is “alive” or “dead” at each time point.
• Plan schedule for the next day - someone will need to check oysters in the morning, mid day, and before the end of the day.

Tuesday Dec 9

Today is the start of survival trials! Here are the things to do:

• Fill each cup with about 12-14 oz of water (enough to fully submerge the oyster but not too full that the cups spill when moved)
• Place individual oysters in individual cups. Record metadata as you load the cups. Record the bag/group the oyster came from and which temperature it is going to.
• Place n=10 oysters from each bag at control temperature (on the plastic table next to the large incubator).
• Place n=20 oysters from each bag at high temperature (inside the large incubator set at 37-38°C).
• Randomize the location of the cups in the incubator so that the groups are distributed between different shelves.
• Record the time oysters were added into the incubator on the data sheet. This is the first time point. Record “alive” for every oyster for this time point.
• Check the oysters again at mid day (about 3-4 hours after the start). Record whether each oyster is alive or dead at this time point. Record the time. Record all data on the data sheet.
• Record the temperature in 5 cups in the incubator and at the control table. Write down these measurements on the back of the data seet.
• Check the oysters again at the end of the day (about 6-7 hours after the start). Record whether each oyster is alive or dead at this time point. Record the time. Record all data on the data sheet.
• Record the temperature in 5 cups in the incubator and at the control table. Write down these measurements on the back of the data seet.

Wednesday Dec 10

Ariana will be on campus today to help. Here are the things to do:

• Continue survival measurements as done on Tuesday (replicate the same procedure recording the new time each measurement is done at morning, mid day, and before end of day.
• Ariana will prepare supplies for sampling (control oysters only) and resazurin measurements (control oysters only)
• Ariana will run resazurin trials on gill tissue of control oysters if there is time
• Ariana will coordinate sampling of oyster tissue if survival trials are nearing completion (nearing mortality targets). This will include labeling tubes and dissecting control oysters.
• If resazurin trials and dissections are done on Wednesday, Ariana will need 1-2 people to assist.

Thursday Dec 11

Ariana will run resazurin trials on gill tissue of control oysters if more trials need to be conducted. Ariana will also coordinate sampling of oyster tissue if this is continued. 1-2 people to assist would be appreciated if sampling is not completed on Wednesday.

Friday Dec 12

Today is experiment clean up! This includes rinsing cups, disposing of oysters, cleaning/feeding tanks, cleaning resazurin plates, or any other clean up needed.

Sample size and groups to transport

We have the following treatments at Westcott:

• “Daily” hardening - control and treated
• “Weekly” hardening - control and treated

In the field, we have seen lower growth in the treated oysters from both hardening efforts.

In order to conduct survival testing, we will do a reciprocal exposure of each group to high and control tempratures for 48-72 h. Therefore, we need enough samples of each group to do these measurements.

Sample size: n=30 oysters from each group

This will provide us with n=10 at control and n=20 at high for each group. In total we will transport 120 oysters. We rarely observe mortality in controls in these tests, so that is why sample size will be smaller at control than high.

Treatments

Reciprocal exposure of each treatment group to either:

• Control (room temperature, ~20°C)
• High (in large incubator, ~38°C)

Treatments will continue until significant mortality (<30% survival at high temperature) is reached, which will likely be within 48-72 h from our previous tests.

Oysters will be placed in individual cups or bowls with seawater and placed in the incubator during the tests. Water changes can be performed every 24 h using water that is pre warmed in the incubator.

Measurements

1. Survival: We will measure survival of each oyster as “alive” or “dead” at multiple time points over 2-3 days
2. Tissue samples: In all control oysters and oysters that survive the stress trial, we will sample gill tissue and freeze for glycogen assays or other testing.
3. Resazurin potential: Depending on results from a trial before the sampling, we may run resazurin on a subset of gill tissue. Ariana will determine this closer to time.

I have premade a spreadsheet on GitHub.

The format will look something like this:

date	site	group	temperature	oyster_id	0	4	8	24	28	32	48	52	56	tube_id
20251209	Westcott	Daily Treated	control	1	alive	alive	alive	alive	alive	alive	alive	alive	alive	1
20251209	Westcott	Daily Control	high	2	alive	alive	alive	alive	dead	dead	dead	dead	dead	NA
20251209	Westcott	Weekly Treated	high	3	alive	alive	alive	alive	alive	alive	alive	alive	alive	3

Sampling

After the stress trial is complete, we will sample gill tissue from all remaining live oysters. This will likely be all control oysters as well as a subset of oysters that survived the high temperature exposure.

• Oysters will be removed from the cup, opened, and gill tissue will be cut.
• Tissue will be placed in snap cap tubes and frozen either on dry ice or liquid nitrogen
• Label tubes with date, “Westcott”, and a running number
• Record the oyster cup number that goes into each tube. We will use this to track metadata
• Place in a freezer box in the -80°C freezer

Pending resazurin results, we may also use some gill tissue from these issues to run resazurin trials. Ariana will plan this out based on how trials go on Nov 26th.

Full protocol

Survial trials:

• Remove oysters from tank
• Place individual oysters into labeled numbered cups
• Fill cups with freshly made instant ocean seawater (ambient temperature) at salinity 20-25 psu
• Record the treatment group that corresponds to each cup number and write a datasheet that has columns for “cup”, “group”, “temperature treatment”, and columns for each time measurement (e.g., 0, 4, 8, 24, 28, 32, 48, 52, 56 hrs)
• Place n=10 oysters from each group on the counter at room temperature (control)
• Place n=20 oysters from each group in the incubator set at 38°C (high)
• Record “alive” for all oysters at Time 0 (when the incubation starts) and record the time oysters were added to the cups.
• Use a temperature probe to record the temperature in n=5 control and n=5 high cups
• About 4 hours later, look at all the oysters and record if they are alive or dead. Record “alive” or “dead” for each oyster on the data sheet under a column labeled “4 hours”
• Repeat at 8 hours later (end of Day 1)
• Repeat this process for Day 2 - record survival for each oyster in the morning, mid-day, and evening under columns for the appropriate time period. At each assessment record the time and temperature measurements.
• Once survival at high treatment is at about 30% or below, the trial can be stopped.

Sampling:

• Remove oyster from cups, open, and take a sample fo gill tissue.
• Place tissue in snap cap tubes and freeze either on dry ice or in liquid nitrogen
• Label tubes with date, “Westcott”, and the cup number of the oyster (e.g., 20251210 Westcott 1, 20251210 Westcott 2, etc)
• Record the oyster cup number that goes into each tube. We will use this to track metadata
• Place in a freezer box in the -80°C freezer

Resazurin:

• If we proceed with resazurin, we will use an additional sample of gill tissue from control oysters to run in resazurin plates at control and high temperatures for each individual
• This would include n=1 sample at high and n=1 sample at control for each oyster, totaling n=10 per group per temperature
• Ariana will run these on Dec 10-11

To do at Westcott & supplies

• Conduct final assessment of survival
• Collect a sample of n=30 oysters from each group
• Add oysters to labeled seed bags with group informatin
• Transport to UW and place in tanks in FTR

To do at UW & supplies

• Prepare batches of Instant Ocean seawater (freshly made) and place in incubator and at room temp to use for water changes
• Prepare tubes, freezer boxes
• Prepare cups and counter space/incubator space
• Prepare dissection tools for sampling
• Conduct resazurin trial on Nov 26th