Yaamini’s Notebook: Gigas Larvae Day 2

A bit of a rough start today

There was a miscommunciation about our use of the reservoir tank near the OA system, so the heaters in our tank were unplugged last night. I turned them back on when I arrived and added an additional heater from the oyster totes since they were at temperature. Stuart also metioned that this water may be super saturated with gasses since it’s not passing through a degassing column. I added an airstone to the tank to let it aerate better. I then took two more heaters and placed them in a 100 L tank and 200 L tank with water so the water would heat faster. Since I had to wait for the temperature in the heated reservoir to reach 20 ºC so I could use it to screen larvae, I took the HOBOs from Laura’s oysters, cleaned them and placed them in my larval buckets. While I was waiting, I saw that the temperature in some of my tanks had dropped down to 18 ºC. I wasn’t sure how accurate the temperature gun was, so I added in another thermometer to a tote without a heater, and closed the door of the SeaLab to retain heat in the room. My guess was that the outside air temperature was affecting the tank temperature. I may need to take the time to insulate the totes. To bring things back to temperature quiclky, I changed the heater setpoint to 28ºC for a brief period of time.

Once the reservoir water hit 20ºC, we started screening! We filled buckets with water from the 100 L tank, since it was at 24ºC. We then would move water at 19ºC from the 200 L tank to the 100 L tank so it could warm faster. Because those tanks had water with a lot of filth through it, we poured the water through a 100 L screen before putting it in the larval bucket. We used a 48 micron screen to catch larvae, then placed it in a tripour with 500 mL of seawater. I sampled 300 µL of the larvae for counting and imaging in triplicate and placed it in a well-plate. I fixed the oysters with Lugol’s and put the plates in the fridge for tomorrow. I also took 300 µL of sample and placed it in an eppendorf tube for either epigenetic or proteomic testing. I pipetted an 80% ethanol solution into the tubes, centrifuged the sample and ethanol, then pipetted out supernatant to rinse out any salts. There was some white stuff at the bottom of my tubes, so I hope this is actually larvae. I need to talk to Laura to see if this was the way she did it, or if she had a more efficient system.

Finally, I fed the oysters a mix of C.iso, Chagra and 609. I topped off the volume in my buckets before I fed, which was a mistake! I forgot to account of the additional volume from feeding — something I won’t do again. Because adding in food made most of the water levels pass the holes in the buckets, I quickly added in tubes, clamps and elbows to prevent water and larvae from flowing out of the bucket.

For tomorrow:

  • Feed
  • Image oysters
  • Ask Joth about heater
  • Move AVTECH to SeaLab
  • Start setting up OA systems
  • Start scoping out setting tanks

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Yaamini’s Notebook: August Goals

I only have five goals

…and I think I can accomplish them! But first, a July Goals recap.

August Goals:

  • Keep larvae alive
  • Analyze SRM data
  • Schedule first committee meeting
  • Finalize proposal and plan of study
  • Talk to Steven about funding after Summer 2018

And who knows? Maybe I’ll have time to do the metaanalysis when the larvae are grown and I don’t have anymore mass spectrometer data to play with. A girl can only hope.

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Yaamini’s Notebook: Gigas Larvae Day 1

I’m the Mother of Oysters

When I first came in, I used PSRF’s temperature gun to check the temperature of each bucket (found here). I noticed that there was some temperature variation between totes, and that the totes in the middle seem to be holding temperature better than those on the edge. I changed the setpoints in all totes with more than one bucket (i.e. every tote except the far left one that I designated Tote 1) to 25 ºC s that the water temperature says around 23-24 ºC. I noticed there was some evaporation from the buckets, so I added a bit more water to each bucket.

I then fed the oysters C.iso with Ashley. I’m keeping track of my feeding here. After using the hemocytometer to count algae, I added half of the needed amount of algae to the buckets and letting it set for a bit. This way, I would give the larvae a chance to feed before saturating the tank some more. I also realized that I severely underfed my oysters on Sunday — they only got one-tenth of the food they should! Not sure why Rhonda and I messed up that calculation, but at this phase they don’t eat too much so I don’t think I’ll suffer a super large mortality.

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Figure 1. Buckets after addition of half of algae quantity needed.

Since Joth was here today, I talked to him about some basic larval rearing things. He advised that I feed either C.iso or T.iso, 609 (Pavlova) and another diatom every day. We also talked about using epinephrine to induce setting as single oysters. He’s going to email me the protocol so I can order the epinephrine. This is something I’ll do when oysters are holding on a 180 micron screen. Joth also said I can use his tanks outside to set up a system of downwellers and upwellers for oyster post-set.

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Yaamini’s Notebook: Gigas Larvae Day 0

Spoiler: We have baby oysters!

I successfully spawned my Pacific oysters this weekend! A huge shoutout to everyone who helped me over the past two days: Rhonda (#TheRealMVP), Steven, Kelsey, Ashley, Megan and Laura.

The relevant data and calculations from my spawn can be found here.

Saturday, July 29

Step 1: Shuck and sex all oysters

The first thing we did was open up all of the oysters and put them into individual paper boats. This was to keep each oyster separate and avoid contamination. We kept all oysters that looked ripe. Anything that looked a little questionable was kept just in case.

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Figures 1-2. Rhonda, Ashley and Megan shucking open oysters.

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Figures 3-7. Examples of ripe oysters.

Using a capillary tube, I took a bit of gonad sample and examined it under the microscope.

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Figure 8. Kelsey adding a capillary tube to a shucked oyster.

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Figure 9. Me sexing an oyster. #ActionShot

It was easy to identify which ones were male and female! Males had active sperm, meaning that they were small and swimming around. Eggs do not have a round shape before being in water, but they look like small seeds.

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Figure 10. Sperm under 10x magnification.

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Figure 11. Eggs under 10x magnification.

Step 2: Create cross matrix

Once we sexed all the oysters, we had to figure out how many crosses we could do. We only had 14 male oysters from all OA tanks, and 2 from the heat shock tanks. To fit the number of buckets we had, we decided to collapse all of the females into pools by treatment, so there would be three egg pools total: one low pH female pool, one ambient pH female pool, and one heat shock female pool. We could then cross each of the OA males with the low and ambient pools, giving us 28 crosses. Replicating these crosses in two buckets would give 56 buckets. Doing the same for the heat shock oysters, we had two crosses with two replicates, so four additional buckets.

Step 3: Strip all gonads

Once we knew what our crosses were, we took each oyster and stripped the gonad. Using a sharp pointy blade, we scored the back and front of the gonad. Then, we scooped the gonad into a pre-labelled tripour. For the females, we stripped 1 gram of gonad per oyster to create our pools. Two of the males ended up not being ripe, so we threw those out and did 52 total crosses instead.

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Figures 12-13. Scoring and scooping the gonad into a tripour.

Step 4: Prepare eggs and sperm for fertilization

  • Screen sperm on an 80 micron screen and catch on a 20 micron
  • Screen eggs on 80 micron screen and catch on 20 micron
  • Combine all relevant eggs for each pool
  • Hydrate eggs for 45 minutes
  • Count eggs in each pool
  • Calculate the number of eggs needed to fertilize each cross

Step 5: Fertilize!

  • Add calculated amount of eggs to a tripour for each cross
  • Add sperm
  • Let eggs fertilize for 20 minutes
  • Check for polar bodies to confirm fertilization

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Figure 14. Polar body on one egg.

Step 6: Distribute fertilized eggs to 5 gallon buckets

  • Buckets should hold at 23 ºC
  • Water bath should hover around 23-24 ºC

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Figures 15-16. Static system pictures post-fertilization.

Sunday, July 30

Step 1: Check developmental stage

When we first got to the hatchery, the larvae were trocophores. This could be because we only fertilized around 6 p.m., and they require about 18 hours to become D-hinge. We only want to handle D-hinge since they hold on a 48 micron screen, and we would be able to account for slower-developing larvae.

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Figure 17. Trocophore larvae around 9 a.m.

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Figures 18-19. D-hinge larvae around 11:30 a.m.

Step 2: Screen D-hinge

While screening, we pooled replicates together to reduce the number of counts we had to do.

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Figure 20. Kelsey screening larvae!

Step 3: Count D-hinge

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Figures 21-22. Ashley dispensing larvae to a Sedgewick Rafter slide, and Laura counting larvae.

Step 4: Redistribute D-hinge to static system

  • Sum total D-hinge per treatment pool
  • Calculate amount of D-hinge to put in each bucket with a stocking density of 4 larvae/mL
  • Put larvae in static system buckets
    • 8 totes, 3 buckets each
    • One tote had a crack, so I had to split the buckets into two separate smaller black totes
  • Fed 60 mL of C.iso and 609 to each bucket

Other notes

While opening the gonad of two female oysters, I found some weird red polychaete thing. I fixed these in 80% ethanol and sent these pictures to Chelsea Wood in case she wanted them!

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Figures 23-24. Weird polychaete things found in oyster gonads.

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