This post details sampling on 28 February 2024 at Point Whitney.

Overview

Today we sampled all lifestages from the lifestage carryover project! This post details our sampling.

Team: Ariana, Steven, Eric, Kathleen, Grace L.

See previous posts on this project in my notebook here and Eric’s notebook here.

The GitHub repository for this project is here.

We are using these metadata sheets today for sampling.

Treatments and protocols

When we first arrived, we set up equipment and talked about the plan for the day. We will have 4-5 people sampling and decided to have 3 people sampling (Eric, Grace, and Kathleen) and 1 person doing the treatments and allocating oysters (Ariana). Steven configured the Apex system, talked with Matt about oyster seed for the next set of experiments, and configured tank equipment.

Our goal today is to condeuct RNA, DNA, and physiology/metabolomic sampling to examine stress responses across lifestages in oysters and evaluate whether thermal preconditioning alters stress responses.

Treatment groups and stock information

Our treatment groups will be as follows.

Lifestages:

• Spat
• Seed
• Juvenile
• Adult

Conditioning treatment groups:

• Control
• Treated

Acute stress treatment groups:

• Ambient
• High

This generates four treatment group combinations for each lifestage:

• Control - Ambient
• Control - High
• Treated - Ambient
• Treated - High

We also have four families for adults (“Bird”, “Blue”, “Orange”, “Pink”). We do not know whether these families are diploid or triploid. Spat and seed are from the same original stock from Oregon and the Juveniles origin is unclear. We need to confirm all stock information.

Temperature treatments

The conditioning treatment refers to an October temperature increase in the “treated” oysters in which oysters were exposed to 25°C daily temperatures (6h) for 14 days.

During this sampling, we are going to conduct an acute heat stress in a reciprocal exposure design. For those exposed to high temperature stress (“high”) we will submerge them in 32°C seawater for 30 minutes. Ambient individuals will be collected from current ambient conditions (13-14°C). This is a +18°C stress. Because of the high degree of stress, it needs to be short term.

We set this up by heating water in one large white tank with all oysters in the other large white tank at Point Whitney. Before exposures, water was heated using 3 800W Finnex titanium heaters on thermostats. We successfully reached 32°C by preheating the water from Monday to 28°C. Today we started the heaters to 32°C at 8am and water was at temperature by 9am.

Oysters were exposed in closed system tubes to keep all exposures independent.

Sampling protocols

We will conduct thermal exposures in a staggered manner to allow for continual sampling and ensure that all groups are treated and sampled in the same amount of time.

We will add a group to the temperature bath, then sample an ambient group. By the time this is completed, we will sample the high temperature group and continue like this until all sampling is completed. We will sample in lifestage groups and take breaks between lifestages.

This worked well today! It took us about 1 hour to sample each life stage group. Each group contained the four treatment groups listed above.

For our sampling, we are taking samples for 3 sample types:

• RNA (tissue preserved in 750uL RNA later)
• DNA (tissue preserved in 70% ethanol)
• Physiology/molecular (tissue snap frozen in tubes for physiology, enzyme assays, and/or metabolomics)

RNA and DNA tubes were preserved in snap cap tubes and phys/metabolomic samples were preserved in screw cap tubes.

During sampling, equipment was sterilized between each sample using 70% ethanol, 10% bleach, and fresh water. We designated people to either shuck oysters, add tissue to tubes, and record data. This way, those handling samples could ensure sterilization and reduce any risk of contamination between samples. Samplers wore gloves. We used tweezers, shucking knives, and tweezers to handle tissue. Specific notes for each lifestage are listed below.

Tubes were prefilled with buffers as needed. Samples that were snap frozen were collected and individually immediately snap frozen.

Sampling spat

We sampled spat first. These are very small (<15mm length) and are not possible to dissect to sample tissue without the shell. For all samples, we added a single spat to each tube and crushed the shell in the tube to allow buffer to preserve the tissue. Therefore, all tubes have tissue from one whole organism and there are no replicate samples per individual.

We took the following samples:

• n=6 DNA samples per treatment group (24 total DNA samples) - 70% ethanol
• n=6 RNA samples per treatment group (24 total RNA samples) - RNA later
• n=5 physiology samples per treatment group (20 total physiology samples) - snap frozen

Therefore, 17 individuals (6+6+5) were added to high stress from treated and control conditioning treatment spat and another 17 were sampled from each group at ambient temperature.

We sampled in this order:

• “Left-Spat” (treated - ambient)
• “Left-Spat” (treated - high) [exposure 09:48-10:18]
• “Right-Spat” (control - ambient)
• “Right-Spat” (control - high) [exposure 10:03-10:33]

All data was recorded in spreadsheets linked below.

A sample of some spat were taken to UW to place in tanks and use for future assay testing. There are probably >50 spat of each conditioning treatment remaining at Point Whitney.

Sampling seed

Seed were sampled in the same way as described for spat but with one key difference. Seed were larger and so we cracked and removed the shell to preserve the whole animal tissue. We still sampled one organism per tube, as they were not big enough for replicate samples.

We sampled at the same replication as described for spat.

We sampled in this order:

• “Right-Seed” (control - ambient)
• “Right-Seed” (control - high) [exposure 10:50-11:20]
• “Left-Seed” (treated - ambient)
• “Left-Seed” (treated - high) [exposure 11:39-12:09]

We had two seed holding containers in each conditioning treatment. These were pooled and then allocated for this sampling.

A sample of some seed were taken to UW to place in tanks and use for future assay testing. There are probably ~10-20 seed of each conditioning treatment remaining at Point Whitney.

Sampling juveniles

Seed were sampled in the same way as described for seed with separate animals for every sample because some were too small for replicate samples. We removed the shell and took samples of the gill tissue for all samples. We are most interested in the gill tissue because we want to know about metabolic stress response and respiratory demands.

We sampled at the same replication as described for seed.

We sampled in this order:

• “Left-Juvenile” (treated - ambient)
• “Left-Juvenile” (treated - high) [exposure 12:55-13:25]
• “Right-Juvenile” (control - ambient)
• “Right-Juvenile” (control - high) [exposure 13:20-13:50]

We had four juvenile holding containers in each conditioning treatment. These were pooled and then allocated for this sampling.

Remaining juveniles were taken to UW to place in tanks and use for future assay testing.

Sampling adults

We decided to fully sample one family for this sampling. We are not able to take samples of all families today and we want to sample a high enough replication to sample individual variation. Therefore, we selected the “pink” family at random. This family had 16 individuals from treated and 16 individuals from control. We split this with 8 animals into ambient and 8 animals into stress.

Each animal was sampled for all assays, so DNA, RNA, and physiology/metabolomic samples are from gill tissue of the same animal.

We took the following samples:

• n=8 DNA samples per treatment group (32 total DNA samples) - 70% ethanol
• n=8 RNA samples per treatment group (32 total RNA samples) - RNA later
• n=8 physiology samples per treatment group (32 total physiology samples) - snap frozen
• n=32 total animals samples

We sampled in this order:

• “Pink-Control” (control - ambient)
• “Pink-Control” (control - high) [exposure 14:30-15:00]
• “Pink-Treated” (treated - ambient)
• “Pink-Treated” (treated - high) [exposure 14:55-15:25]

Bird, Blue, and Orange adults were not used in this sampling and are still at Point Whitney.

Sampling finished and we left at about 16:30.

Data upload

Metadata for sampling was typed from physical notebook pages and stored on GitHub here.

Sample storage

Kathleen, Grace, and Eric took samples and extra oysters back to UW after sampling. RNA later will be left at room temperature until Thursday PM or Friday AM and will then be put in the -20°C freezer. DNA samples will be added to the 4°C fridge. Physiology/molecular samples will be added to the -80°C fridge. Boxes will have the date (20240228), “Huffmyer”, “LCO-24”, and the sample type labeled.

We will next work on using extra oysters to trial assays and processing samples after we look at Eric’s qPCR results from his November sampling.

Here are the storage notes from Eric:

RNA: was stored at room temp overnight on feb 28. Then moved to 4º in FTR 213 at 11 AM on feb 29. Tomorrow (feb 30) around noon I will remove the RNA later from tubes and store in -80. This is following their recommendation.

DNA: stored in 4º in FTR 213 at 7:30 pm, feb 28

Phys: taken from dry shipper, put into boxes, immediately put in -80º at 7:30 on feb 28. They are not in the leftmost -80 freezer because that one is completely full. They are in the freezer to the right of it, on the top shelf.

All are in clearly labeled boxes e.g 20240228 LCO-24 DNA AHuffmyer or EE

Pictures

Here are some pictures of the day taken by Kathleen!