This post details sampling for 10K seed hardening project at UW.

I started a GitHub repo for this project (will add more info to it as we go) and is available at this link.

Overview

In this project, we conducted hardening treatments on seed from Point Whitney. We had n=2 bags of 1,000 seed each exposed to each of 5 treatments:

• Control (no treatment)
• Fresh water (1 h exposure)
• Fresh water heated to 35°C (1 h exposure)
• PolyIC (3 h exposure)
• 35°C seawater (1 h exposure)

These treatments occurred for 3 weeks, 3 times per week at Point Whitney.

A subset of oysters (n=150 from each bag) were transported to UW for samping and testing. The remaining oysters are left at Point Whitney in the lagoon off the dock at their normal location.

Based on the results of sampling, we will decide either to do another round of hardening treatment or proceed directly with outplanting.

The goal of sampling is to conduct the following analyses/response variables:

• Survival
• Resazurin (metabolic rate)
• Na/K ATPase enzymatic activity
• Citrate synthase activity
• Lactate dehydrogenase activity
• RNA sampling
• DNA sampling
• Glycogen content

Note that I’ll add more details here as we progress through sampling and add data to the GitHub repo.

Monday July 22

Preparing supplies for sampling

Today I prepared supplies for sampling. I labeled tubes and prepared reagents for sampling. The plan for sampling is as follows below. I tested dissections for these seed and because they are small, tissue samples will be from gill/mantle because they are too small to reliably separate the gill tissue.

1. RNA: Sample oyster gill/mantle tissue in n=5 oysters per bag (n=10 oysters per treatment) in response to 1 h exposure to 18°C and 42°C. Sample with RNAlater into tubes and store at 4°C for 24 h. Remove RNAlater after 24 h and store at -80°C. Tubes R1-R100.
2. DNA: Sample DNA from oyster gill/mantle tissue in n=5 oysters per bag (n=10 oysters per treatment) from the ambient condition they are in. Sample into 70% ethanol and storing at 4°C. Tubes D1-D50.
3. Citate synthase & Lactate dehydrogenase: Sample oyster gill/mantle tissue in n=5 oysters per bag (n=10 oysters per treatment) in response to 1 h exposure to 18°C and 42°C. Sample by snap freezing in liquid nitrogen in tubes and storing at -80°C. Tubes E1-E100.
4. ATPase: Sample oyster gill/mantle tissue in n=5 oysters per bag (n=10 oysters per treatment) in response to 1 h exposure to 18°C and 42°C. Sample in SEI buffer and snap freezing in tubes and storing at -80°C. Tubes A1-A100.
5. Glycogen: Sample whole organism tissue from n=5 oysters per bag (n=10 oysters per treatment) from the ambient condition they are in. Sample by snap freezing in tubes and storing at -80°C. Tubes G1-G50.
6. Resazurin: Measure metabolism via resazurin with Colby’s protocols in 4 h exposure at 18°C and 42°C in n=20 oysters per bag (n=40 oysters per treatment). Conduct measurements with treatment randomized across time. One bag measured each day. Followed by imaging all oysters for size for normalization and track survival after measurements for 24 h. Survival tracked by rinsing cups and refilling with new seawater and left in the cold room at 13°C for 24 h. Will be led by Colby.
7. Survival: Measure survival along with resazurin as described above. Also measure survival with n=40-50 oysters per bag (n=80-100 oysters per treatment) with exposure to 2-3 h exposure to 42°C or 18°C. Alternative is to hold at 18°C or 42°C and analyze time to mortality. We will think about this pending resazurin results.

Resazurin and survival

Today we tested resazurin on one randomly selected bag (bag 47, 35°C hardened group). Oysters were put in plastic cups in 30 mL of resazurin. The incubator was set at 42°C. Colby took measurements at the start and then every 30 minutes for 4 hours. Here are some things we learned and altered in the protocol:

• We decided to lower the volume to 20 mL. The range of values was lower than Colby has seen in previous runs and because the oysters are small, 20 mL will be more appropriate.
• We added a rinsing step to clean the 96 well plate between every run.
• We measured survival after the run and found that within 24 h, 2 control oysters and 3 high temperature oysters died. This was lower mortality than expected. We will measure control oysters tomorrow to see if this is a hardening effect.
• The data was a bit noisy - we think this could be because measurements are being taken too frequently, leading the oyster water to cool during measurement. We will now measure every 1 h.
• We did not see any difference in rates between high and control temperatures, which is contrary to the expectation from what Colby has seen before. This could also be an effect of hardening treatment, so we need to measure a control group and measure this same group again once protocol adjustments are made.

We took photos for size and Colby is keeping notes in his notebook.

Tuesday July 23

Sampling RNA

Today I conducted a temperature experiment to sample RNA in response to temperature stress in all hardening treatments. Here is the protocol that I used. All detailed notes are in my notebook and I’ll add data tables and notebook images to the GitHub early next week.

1. Obtain n=10 oysters per bag. Preheat seawater to 18°C and 42°C. Label 6-well plates with 1-5 in 5 of the wells and label the top of the plate with the bag number and either control or high treatment.
2. Once water is ready, fill 6 well plates (16 mL) and add one oyster to each well. Take an image of all oysters in their assigned well with a scale bar to correlate response to oyster size.
3. Place the control plate on the counter at 18°C and the high plate in incubator at 42°C. Temperature loggers are already deployed in all locations.
4. Stagger start times for each control and high pair of plates for each bag by 10-15 minutes to allow me to sample at exactly 1 h exposure for all plates.
5. After 1 hour, sample oysters by taking each oyster out of the plate, open the oyster, and take sample of mantle/gill tissue using tweezers and razor. Clean tools with ethanol (70%), 10% bleach, and DI water between samples.
6. Put tissue in tube with 750uL RNAlater. Move onto the next plate at 1 h exposure. Repeat until all plates are done.

Sampled all tubes, no mortality observed. Tubes kept at 4°C and plates cleaned. Kept in 2 boxes labeled with 10K seed and date.

Survival

I checked mortality of yesterday’s resazurin samples. There was no new mortality from yesterday, totaling 2 dead in control and 3 dead in high temperature. I cleaned up all cups.

Wednesday July 24

Sampling glycogen

I sampled for glycogen content by taking n=5 oysters per bag, removing all tissue, and adding to a tube. Tubes were snap frozen in liquid nitrogen and stored at -80°C. Due to small size, I kept all tissue. In the future we can try sampling specific tissues. Sampled with cleaning as described during RNA sampling. Kept in box in middle -80°C on fourth shelf on the right, labeled with 10K seed and date.

Sampling DNA

I also sampled for DNA by taking n=5 oysters per bag, removing gill/mantle tissue, and adding to a tube with 70% ethanol. Tubes were kept at 4°C. Sampled with cleaning as described during RNA sampling. Kathleen assisted. Kept in 1 box in 4°C in FTR 213, labeled with 10K seed and date.

Processing RNA samples

In the afternoon I removed RNAlater and stored samples at -80°C.

Kept in 2 boxes labeled with 10K seed and date in middle -80°C in fourth shelf on the right.

Resazurin and survival

Today Colby ran resazurin on Bag 37 (control hardening treatment) using the protocol adjustments listed above. Here is what we noticed:

• In the first 2 h, metabolic rates were higher at 42°C noticably. At this time, 5-6 oysters had already died and these oysters had the highest metabolic rates.
• After 3-4 hours, high temperature rates plateaued and control rates continued to increase, which is interesting. The shape of the curves looked quite different between treatments.
• The dead oysters also showed the slope plateaued.
• We watched mortality for 24 h after and Colby took size images as we did on Monday.
• There was a clear difference in these results as compared to Monday! We will process another bag tomorrow.

Thursday July 25

Resazurin and survival

Colby noticed that all but 2 oysters were dead this morning from the 42°C resazurin treatment! Only 5 of the control died, indicating there may be some effect of resazurin and/or small water volume in control oysters.

Today Colby ran resazurin and survival for a fresh water treated bag. Notes are in his notebook and he is saving the data.

Remaining work

Next week, I’ll sample for enzymes and ATPase and work on more survival measurements. Colby will continue resazurin measurements.

Friday July 26

Resazurin and survival

Colby followed up with survival monitoring of the resazurin batch from 7/25. All notes are in Colby’s notebook. I’ll update the notes here after sampling is completed and notebooks are collected.

Other notes

There was an accidental fresh water stress sometime in the evening of 7/26 to the morning of 7/27. Kathleen caught this issue in the morning of 7/27. Water was leaking from the ceiling.

Kathleen returned conditions to normal late morning on 7/27.

Monday July 29

Resazurin and survival

Today Colby ran resazurin and survival on the control group of seed (Bag #37). We chose to do controls again to see if the fresh water exposure over the weekend altered responses. We saw consistent responses with last week. We saw mortality in high temperature oysters (12 total) and that the oysters that died had the highest metabolic rates. The rates peaked early in high temperature oysters, leveling off at about half way through the incubation. The control temperature oysters increased more slowly and continued to increase throughout the entire exposure. We are seeing these same patterns as we did last week. Given the consistency, we will proceed with treated oysters the rest of the week.

I’ll check survival in the morning.

Sampling for enzyme activity

Today, I sampled oysters for enzyme activity (citrate synthase, lactate dehydrogenase, and ATPase). To do this, I exposed n=5 oysters per bag to 42°C and n=5 oysters per bag to 18°C (totaling n=10 oysters per treatment to each temperature). This was run in the same method as done for RNA sampling last week. I staggered start times by 20-30 min intervals to allow for sampling for 1 h exposure for all oysters.

At sampling, I dissected the gill/mantle tissue as done for prior sampling for RNA and then split the tissue in two. One tissue sample went into a tube labeled with “A” and a running number (A1-A100) with 500 uL of SEI buffer for ATPase activity. This was then snap frozen in liquid nitrogen and stored at -80°C.

The second tissue sample was put into a tube labeled with “E” and a running number (E1-E100) and flash frozen and stored at -80°C. This will be used for citrate synthase and lactate dehydrogenase. I’ll likely have to optimize these assays for lower tissue input due to the small size of oysters.

This took place form about 11:00-17:00. Samples were kept in 2 E boxes and 2 A boxes in the middle freezer, fourth shelf, on the right. I’ll organize the freezer tomorrow.

Tank maintenance

I added about 15 mL of food to the tank at 09:00. I took probe measurements for temp, pH, and salinity at 17:00.

Tuesday July 30

Resazurin survival

Today I checked survival for resazurin. There are 3 dead in control and 18 dead in the 42°C group. I uploaded all survival data to the GitHub today and asked Colby to upload data to GitHub. Survival was the same at 11:00 and 15:00.

Tank maintenance

I did a 75% water change today and adjusted salinity to ~26 psu. I fed about 20 mL of shellfish food (Reed Mariculture instant algae #1880) at 11:30.

Data uploading

I uploaded images, notebook images, and available data to GitHub today. The 10K seed repository is here.

Planning next week

Here is the order of resazurin/survival for this week:

• Tomorrow (Wed 7/31): Bag 30 (temperature hardened)
• Thursday (8/1): Bag 75 (fresh water and temperature hardened)
• Friday: Montior survival
• Monday (8/5): Bag 50 (immune hardened)
• Tuesday (8/6): Monitor survival
• Wednesday (8/7): Bag 47 (temperature hardened)
• Thursday (8/8): Bag 56 (fresh water hardened)
• Friday (8/9): Monitor survival
• Monday (8/12): Bag 36 (control)
• Tuesday (8/13): Monitor survival
• Wednesday (8/14): Bag 49 (immune hardened)
• Thursday (8/15): Bag 76 (fresh water and temperatured hardened)

Wednesday July 31

Resazurin completed on Bag 30 (temperature hardened). Detailed in Colby’s notebook.

Thursday Aug 1

Resazurin completed on Bag 75 (fresh water + temperature hardened). Detailed in Colby’s notebook.

Friday Aug 2

Survival monitored from Bag 75. Detailed in Colby’s notebook.

Monday Aug 5

Resazurin completed on Bag 50 (immune hardened). Detailed in Colby’s notebook.

Tuesday Aug 6

Survival monitored from Bag 50. Detailed in Colby’s notebook.

Wednesday Aug 7

Resazurin completed on Bag 47 (temperature hardened). Detailed in Colby’s notebook.