Grace’s Notebook: Day 1 of Trial 2 of Geoduck Stripspawn experiment – KCl dose + duration

Today was Day 1 of the second trial of geoduck stripspawn. We created an experimental design that takes into account both KCl dosage, and duration of the dose exposure. Overall things went pretty well (details in post), and on Wednesday, I’ll check for D-hinge development.

Google doc with plan

(written in prep for the day, things changed and will be noted in this post)

8:30am – Pt Whitney

Started at Pt Whitney, biopsy punching gonad tissue and checking for sex and ripeness.

I started out getting two or three males, and used a new biopsy punch with each geoduck. I placed the biopsied males in the right tank and grabbed new geoduck to biopsy from the left tank.

I then found two females, and then I found one that looked like a hermaphrodite, which was pretty cool! I’ve never seen that before:

img

Then I kept looking and finally found another female, though it may not have been as ripe as the other two. I left a little after 9:45am.

10:15am – Taylor Hatchery

I started out with cleaning the work area, cleaning the 20 20um screened mini-silos, gathering materials. Benoit, Michelle, and Molly helped by getting a filtered saltwater line available for me to use in the work area.

Once I had the filtered saltwater, I started setting up the tripours with the different mM KCl dosages, as well as some next to them full of filtered saltwater. While I was doing this, Benoit was setting up buckets in the bucket room with airstones and water in which the fertilized eggs will grow out in at the end of the dosing and fertilizing.

Setting up the dosages:

I filled the tripours up to 800ml (any more than that and the water will spill over once the 20um silo is placed in). When making the dosed saltwater, I made them as though they’d be filled to 1000ml so that the math would be easier.

I made them in a 3L pitcher, so I did them in sets of two. Example: to make the 20mM, I placed 20mL 2M KCl and 1960ml filtered saltwater. I then dispersed it into two tripours, up to 800ml. I did that again, and then moved on to the next dose.

KCl mM 2 M KCl stock to add (ml) ml saltwater to add
20 10 980
50 25 975
60 30 970
80 40 960

Tripour treatments:
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Check female geoduck ploidy

Before dissecting the females and stripping the eggs, I measured the shells:

female number length width ratio diploid? (yes if ratio < 1.62)
1 127 73 1.739 no
2 121 71 1.704 no
3 124 80 1.55 yes

Benoit ran the ctenidia samples from all three females on the flow (? is that what it’s called) to check for ploidy. All three had the same ploidy, but it isn’t known if they are all three diploid, or all three triploid. Benoit believes they are likely all three diploid.

Stripspawn females

I dry stripped some eggs from all three directly into the screened silo sitting in tripour 19 (50mM with no hydration), and timed for 20 mins.

During those 20 mins, I stripped eggs from the three females into some saltwater. Benoit helped get a lot of eggs out. One female was much more ripe than the other two. Of the remaining two, one female was not very ripe at all unfortunately.

Benoit helped with screening and cleaning the eggs, and counted them using the coulter counter. The first counts showed that there was about 4 million eggs in the 3L pitcher.

from Grace’s Lab Notebook http://bit.ly/2IAGN4o
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