Today we did a broodstock immune priming treatment and spawn with oysters at Point Whitney.

Overview

Today we exposed a set of broodstock to PolyIC immune priming immediately before strip spawning to test cross-generational priming effects. This post details activities from today.

The work today followed the spawning protocols I have described previously.

Broodstock

The broodstock were provided by Point Whitney and are stock that are 2-4 years old from Sequim Bay.

~50-75 broodstock were treated and another set of ~50-75 were not treated and left as control.

Immune Priming

I primed the oysters using PolyIC immersion as we have done in the past and discussed in this post.

Briefly, I dissolved the 50 mg of PolyIC stock in 50 mL of water (1 mg/mL). I targeted a concentration of 0.75 ug/mL for the exposure. The previous work in the posts linked above used 0.38-0.75 ug/mL and found no difference in exposure effects. I went for the higher concentration today. I therefore added ~20 mL (20.25 mg total) of the stock to ~27,000 mL of seawater in a bin. This generated 0.75 ug/mL exposure solution.

I added a bubbler to the bin with treated oysters. I kept the control oysters in a bin with a bubbler but without any PolyIC addition. They were exposed from 08:30-10:30 (2 hours).

Here is an example shot of the oysters.

Treated oyster bags:

Control oyster bags:

Strip spawning

Carrie and I then did a strip spawn! We did the control group first while the treated oysters were in their treatment. Here is our general protocol:

• Open oysters and sex them (male or female).
• Keep high quality gametes (large round eggs, concentrated sperm, low debris).
• Isolate female gonad tissue from selected females in a plastic bag and rub to homogenize. Pour onto a 20 um screen and rinse into a 1 gal bucket. Keep at 25-26°C.
• Take a sample of male sperm from each selected male into a glass dish with some seawater. View under the scope for motility.
• Allow eggs to hydrate for 20-30 min.
• Fertilize with ~10 mL of sperm water into the bucket.
• Pour into large 5K liter tank!

Control = 5K tank #1
Treated = 5K tank #2

All spawning and fertilization occured before 12 noon.

About half the oysters had nice gametes and we kept gametes from 12 males and 14 females from the control group. We kept 14 males and 15 females from the treated group.

Here is an example of males after opening and sexing.

This is what the eggs looked like after taking female gametes and combining.

The gametes selected for fertilization looked similar to the images below.

Eggs:

Sperm:

Next steps

Carrie is going to drop the larvae on Thursday and again on Tuesday of next week. I will go to the hatchery on those days to take samples in RNA/DNA shield of larvae from each treatment at each of these time points.

The larvae will be reared and shipped for setting using Point Whitney’s standard production protocols.

We cracked open a few of the USDA broodstock and didn’t see any gametes.

I also took a few gamete samples in 15 mL tubes and kept in a cooler (not directly on ice) to bring to Mac at UW tomorrow.

10K seed

I also did a 10K seed immune priming exposure (bags 49 and 50) from 10:30-11:30.

Notebook images

Here is an image of Carrie’s notebook. I’ll take pictures of this notebook again on Thursday to see the conditions of the larvae over the next few days.

Here are the notes I took today.