Yaamini’s Notebook: Gigas Spawn

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.



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





Figures 3-7. Examples of ripe oysters.

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


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


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.


Figure 10. Sperm under 10x magnification.


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.



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


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



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.

screen shot 2017-07-30 at 10 54 01 pm

Figure 17. Trocophore larvae around 9 a.m.


screen shot 2017-07-30 at 10 54 35 pm

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.


Figure 20. Kelsey screening larvae!

Step 3: Count D-hinge



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!



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

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Yaamini’s Notebook: Manchester Conditioning Update11

Yaamini’s Notebook: Manchester Conditioning Update 10

It’s getting ugly

I had 16 oysters die today from various treatments. Based on Steven’s suggestion on Monday, I moved the oyster bags to individual 5 gallon buckets on Laura’s larval heated line, around 18 ºC. When I opened oysters that were dead, they all looked really ripe, and there were no signs of any disease. My guess is there’s something in the combination of the kiddie pool and high temperature that’s getting to them.

Table 1. Revised oyster counts in each tank, as of July 25.

Tag Label A B Total
1 3 7 10
2 5 8 13
3 2 6 8
4 2 7 9
5 6 8 14
6 4 8 12
Heat Shock 3 4 7
Full Amb 0 6 6
Spare 1 3 4
Total 26 57 83

I cut open the ones that were dead and with the exception of two, preserved them for future testing. Three oysters were stored in falcon tubes with ethanol, three in the freezer and a jar with about six oysters was put in the fridge. Not entirely sure what to do with them yet, but at least I have them. The ones that were died were quite ripe and did not look unhealthy.





Figures 1-4. Examples of oysters that died.

Oysters in Figures 1-4 were also sexed. All were male.





Figures 5-8. Microscope images of male gametes.

Other things I did today:

  • Turned off immersion heaters
  • Cleaned algae header and filled with 1.6 L Reed’s paste
  • Removed lines from oyster tanks and bleached algal line
  • Counted about 30 2 gallon buckets that can be used for fertilization
  • Set up buckets for each bag of oysters with 26 L/hr drippers and airstones


Figure 9. New set-up.

  • Cleaned gigas and counted mortalities
  • Tried measuring pH of kiddie pool water. Both durafets read different pH values for Pool B (farthest from wall).


Figure 10. Durafet monitor readings for Pool B.

  • Put the Heat Shock B oyster we thought had died back into a different bucket since it’s shell was completely closed as we were about to leave.
  • Pumped T.iso in for oysters to feed instead of paste, dosing pump at 50%

Main changes:

  • Each oyster bag has it’s own 5 gallon bucket, increasing flow and reducing density
  • Temperature is now around 18ºC instead of 23ºC. I talked to Rhonda and she’s a bit concerned that the oysters may start to reabsorb their gametes. She once had to drop broodstock to 15ºC two days before a spawn and it was fine. Maybe this will also be okay? Can they really reabsorb that quickly?
  • Oysters are being fed live algae

I will be back Thursday to check on oysters and continue preparing for spawn. I’m really hoping that no more die — I think we’ve eliminated all possible stresses! All I need is for them to be alive until I spawn them.

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Yaamini’s Notebook: Manchester Conditioning Update 9

The mortalities continue

The past few days I’ve seen a string of mortalities, primiarly in conditioning Tank A.

Thursday, July 20

Grace vacuumed the oysters and checked for mortalities while she and Laura were out there. She found three dead oysters: 1 from Tank 1B, 1 from Tank 1A and 1 from Tank 5A. Tank 5A had been dead for a few days, while Tank 1B and 1A were freshly dead.




Figures 1-3. Dead oysters.

Saturday, July 22

Laura cancelled at the last-minute on Saturday, so I took her car and went to Manchester. When I got there, all of her larval buckets were brimming, about to overflow. I replaced the banjos then cleaned the ones I took out. I also checked the heater setpoints.



Figures 4-5. Heater setpoints upon arrival.

I then started on cleaning my oysters. I immediately noticed that the oyster bags themselves were collecting a lot of fecal matter and algae. Most of my time vacuuming was spent vacuuming the bags. Next year, it may be better not to use these bags and opt for something like a tray that can be removed and cleaned. There were three dead oysters: 1 from Full Amb B and 2 from Tank 3A. When I looked at their gonads, they were milky. Even though my oysters have been consistently dying, they’re also consistently ripe. I need to start sexing them soon and checking for active sperm.




Figures 6-8. Gonad development from dead oysters.

Because of the mortalities, I reduced the right heater setpoint.


Figure 9. New heater setpoint.

I continued my cleaning kick and rinsed Laura’s downwellers with freshwater. I then fed her algae tanks a mix of C.iso and Chagra and checked that the dripper was working. I then added 1.6 L of Reed’s paste to my algae header tank and turned off the dosing pump so I could clean the heating tank.

When I opened the algae heating tank, it looked and smelled like a swamp monster graveyard. The entire tank was filled with dense clumps and string of algae. I think algae had been growing (and dying) in the heating tank before being fed to the oysters. My guess is that this was causing some of the oyster deaths.


Figure 10. An example of what was growing inside the heating tank.

As I was cleaning the heating tank, I also wanted to flush algae from the water line. When I increased flow from my heated line, I noticed there was no water. I called Laura and saw that her outside tanks were also getting no water. The water shut off at some point while I was eating lunch, so it wasn’t off for more than an hour. Luckily, Rick stopped by the hatchery at that time. He discovered that the float valve in our seawater reservoir tank in the warehouse had been jammed in a way that prevented water from flowing. He was able unjam it and get water to flow again.

Another thing I noticed on Friday was that the two tanks had a 1.5ºC-2ºC difference in temperature. After cleaning the heating tank and pipes feeding water to the kiddie pools, I visually adjusted flow so they looked equal in both tanks. I checked the AVTECH monitor and saw that temperature in both pools were within 1ºC. I had to catch a ferry and didn’t have time to measure flows. I set the dosing pump to 60% and ran out the door.

Sunday, July 23

Laura went out to Manchester so she took a look at my oysters. The first thing she found was a high number of mortalities: 2 from Tank 3A, 2 from Full Amb A, 1 from Tank 5A, 4 from Tank 4A, and 1 from Heat Shock B. She also saw that the pools were extremely dark when she got there and the food was completely dark. This means that in 24 hours, the oysters were fed 1.6 L of Reed’s paste. It’s possible that they were suffocated by their own lunch.






Figures 11-15. Dead oysters.

I also had her open the oysters that were recently dead.






Figures 16-20. Gonads of recently dead oysters. All are milky and ripe.

The algae heating tank was not dirty, which was good. Because the pools were slimy and filthy, Laura cleaned them. However, this meant that she moved the oysters out of the water. I’d been avoiding this because I was told they would spawn out. Laura, however, did not know this. She adjusted flow to 2 L/min into each kiddie pool and set the dosing rate to 40 after adding 750 mL of food to the algae header. Before she left, she checked to ensure the oysters had not spawned out.

Monday, July 24

No mortalities today! I took care of a couple of others things while I was out after cleaning the kiddie pools and heating tank. I counted about 60-70 tripours and obtained 8 totes that fit 3 buckets each. I need one more tote to fit 25 buckets total.

Table 1. Revised oyster counts in each tank.

Tag Label A B Total
1 6 7 14
2 8 8 16
3 2 6 8
4 2 7 9
5 7 8 15
6 5 8 13
Heat Shock 5 5 10
Full Amb 2 7 9
Spare 2 3 5
Total 39 60 99

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Laura’s Notebook: SRM Dilution Curve Sample Prep

Prepping dilution curve samples

After we run all our samples Yaamini & I are going to run 10 samples with mixtures of geoduck/oyster protines. We will prepare them at different, known preportions of G:O, and see how the peptides we each targeted become more/less prevalent. This is good practice to prove that we are measuring what we say we are.

Yaamini did a great job calculating/writing up the dilution calculations (after a couple iterations, see here and here

As Yaamini did, I pooled 5ul of 5 randomly selected samples for a total of 25ul of pooled sample – I selected samples that had the full 2ug/ul (as opposed to those that had low concentration), pipetted them into a centrifuge tube, vortexed, then spun down gently:

  • 120
  • 47
  • 79
  • 60
  • 9

Then, I got Yaamini’s dilution curve samples, that already have PRTC & ACN+FA, out of the freezer, and pipetted the designated vol of my pooled geoduck sample into each tube:


Then I vortexed each, spun down gently, labeled autsampler vials, and transferred the dilutions into the vials. I put them in the -20 freezer until we are ready to use them.

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Laura’s Notebook: Remaining SRM Timeline

How many samples can I run?

@ 8:30am on Monday 7/24 I got my samples started again. I need to figure out how many more samples I can run within the given timeline. Here are the considerations:

  • Running samples in batches of (5 samples) + (1 QC) + (1 blank) = 500 minutes total, so that’s 100 minutes / sample.
  • We have until 10am on Friday 7/28
  • Need 40 hrs for the dilution curve run
  • So, need to be done with my samples by Wednesday 7/26 @ 6:00pm
  • Time between this morning @ 8:30am when I re-started my samples & 7/26 @ 6pm = 57.5 hrs = 3450 minutes / 100 minutes/sample = 34.5 samples.
  • I have 25 samples left to run, plus 2 blanks (Gblank & OBlank) which I could run twice.
  • Should be done with my samples on Wednesday @ 5:30am, this includes 1 run of each blank.

Thoughts on whether or not to re-make samples

Up to this point I have 20 samples where a handful of peptides don’t show up from both PRTC and my samples. In PRTC there are 4/9 poor quality peptides, and in my samples 3/39 poor quality transitions. In an ideal world I would remake these ~20 samples, run the new batch twice while being careful with freeze/thaw and time out of the freezer. However, I don’t have time for 2 runs of remade samples. I could remake, then do 1 run of each in the hopes of capturing data on those 3 transitions. However, I think it’s best to have replicates of the 36 good quality transitions in my samples. Also, the 3 poor quality transitions were not in the sample proteins, so I can likely draw conclusions about protein quantification from the other 2 transitions. The differing rates of peptide degradation between samples does make me a little concerned; I’m wondering how folks take this into consideration.

Samples left to run:
#### Plate 2 – 23 + 2 blanks | | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |—|——–|——–|——|——|——|——–|——|——| | A | | | | | | | | | | B | G041 | G066 | G105 | G032 | G129 | G054 | G081 | G003 | | C | G074 | G014 | G049 | G053 | G104 | G055 | G042 | G064 | | D | G073 | G057 | G007 | G070 | G001 | G071-B | G062 | | | E | GBlank | OBlank | | | | | | | | F | | | | | | | | |

#### Plate 1 – run 1 – 1 sample I didn’t re-run | | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |—|——|——-|——|——|——|——–|——|——| | A | | | | | | | | | | B | | | | | | | | | | C | | | | | | | | | | D | | | | G128 | | | | | | E | | | | | | | | | | F | | | G122 | | | | | |


  • The following samples were put into the autosampler tray @ the following dates/times:
    • 7/23 @ ~8pm: G041, G066, G105, G032, G129, G054, G081, G003, G074, G014, G049, G064, G073, G007, G001, G071-B, G062.
    • 7/24 @ 2:25pm: G122
    • 7/24 @ Xpm: TBD
    • 7/25 @ Xpm: TBD

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Laura’s Notebook: Mass Spec, good things to know

Pro Tips for using UWPR mass spec

  • On the nanoAquity UPLC Console keep an eye on the Binary Solvent Manager screen to monitor pressure. Right click over the plot area, click Plot Properties, and change the time frame to 10 hrs.
  • If you miss-assign a vial location, or remove a vial too early (like I did), the mass spec will sense this and display an error “missing/wrong plate or vial”. It will stop running. The AutoSampler program will need to be reset, and sometimes also need to hit the manual reset button on the machine. Use the same process as when a program is not responding:
  • If program is not responding, wait until the current sequnce is done, then it may start responding. If still not responding, close the program and re-open. do the following:
    • On Thermo XCaliber go to Status tab on the left side of the screen
    • Check that under Waters nanoACQUITY and TSQ Vantage they both read “ready to download” – if so, you can start your run.
    • If an error, close all programs, reboot PC.
    • Open these 3 windows: Thermo Sequence Setup, Xcaliber, NanoACQUITY UPLC Console, Thermo TSQ Tune Master. It may automatically connect. If not:
    • Hold RESET tiny black push button down for 3 seconds. This should connect the software to Vantage.
    • Before running sequences, on Xcaliber, select Actions -> Automatic Devices On
    • Select & add desired sequence rows
    • Push green “play” button – should work out.
  • If you try to open a raw file in Skyline and it says “permissions denied” or something like that, try closing Skyline and re-opening it.
  • In Thermo Xcaliber Sequence Setup, be careful when modifying the sequence table:
    • If you’ve already added a row to the Acquisition Queue, but you want to change something in that row, DO NOT simply edit then re-add it. Instead:
    • First, delete the rows in the Acquisition Queue that you no longer want by: clicking check box, then hitting the Delete button on a Windows keyboard, or FN+Delete on a mac (if using Team Viewer)
    • Second, start a new row in the sequence table, complete all cells, then add that to the queue.
    • Do not edit then re-add any rows that have already been added to the queue.

Making a method

  • It’s best to work with someone who has used your machince before, get a method from him/her, and tailor it to your needs. Prisca, or someone from the McCoss lab have helped Emma in the past.

Mass Spec Setup tutorial

  • Things that are sitting out in the UWPR mass spec room are things that you can use
  • First thing to make is the pre-column for the trap, we used 4.5cm, with the frit ~3mm. Trap is wider diameter 100um, and different material. Sample first goes on to the pre-column, junk is washed away within the pre-column. When installing: frit points towards the mass spec.
  • Then, go to the nanoQCQUITY; start testing your columns to make sure they can handle the pressure during your sample runs
    • Click on 0.000 uL/min, select “Trapping”
    • Solvent A: 0.3 ul/min, 5% Solvent A
    • Confirm it’s still trapping
    • Watch pressure change on top plot; make sure it doesn’t go too high
    • Change the flow rate on A1 to 0.5 ul/min, after it levels off record pressure
  • Attach analytical column.
    • look at it in the light, make sure there are no bubbles
    • Attach
    • Change solvent B to 5%, analytical, and @ 0.2ul.
    • Keep an eye on the tip of the column; once a droplet begins to form, wait 10 mins. If pressure looks good, then… TB continued…

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Katie Notebook: Results Organized by Population


Now that I officially have all the oysters sampled accounted for, staged, and recorded I split up my results by population as suggested by Laura. I went through and reviewed all my oysters I had previously labeled as HPM or HPF and, based on the literature, (Oates) decided if they were developing hermaphroditic oysters or or actually oysters going towards being male or female (sperm or eggs were degrading). That gave me only three categories to work with and much more visible results. She was right! There do seem to be more differences between populations than between treatments overall. It also seems like overwintering temperature has a bigger impact than OA treatment does, but that is just at first glance. I’m not sure how to pull any significant results from my figures at this point….my statistic skills need some brushing up on.

Check out these results and see if anything should potentially be combined in different ways. The full updated excel document is posted under Histology staging results.

I first made pivot tables to get all the total distributions of sex within each population and treatment:
Screen Shot 2017-07-23 at 9.17.41 AM

Then I organized it all in a new spreadsheet so that figures can be easily generated in the future using any combinations of these results:
Screen Shot 2017-07-23 at 9.18.06 AM

Then here are some figures I made that show the comparison between overwintering temperature results! Hood Canal oysters show a significant different between the two temperature treatments, South Sound oysters are pretty similar within the population across temperature treatments, and then the Fidalgo Bay oysters are interesting in that they have the exact same results, but there are no females…
Screen Shot 2017-07-23 at 9.20.04 AM

I also started play with OA comparison figures. I could do it in two ways: Compare, for example, both the treatments that were overwintered at 10 degrees but had different OA treatments OR I could compare oysters that had the same OA treatment but were overwintered at different temperatures.

For now I am going to work on making a google doc to begin typing up my methods.

Laura’s Notebook: Second SRM sample prep

Readied last 25 peptide samples for mass spec

0) Labeled mini centrifuge tubes with second batch of samples, and reviewed calculated volumes of sample & Final Solvent to add to each: image

1) Borrowed one last 20ul aliquot of PRTC stock from Emma/Jenn – PRO TIP: order mass spec supples way in advance! 2) Added 30ul Final Solvent, vortexted, spun down 3) Pipetted 1.88ul diluted PRTC to centrifuge tubes 4) Pipetted designated volume of Final Solvent, as per my calculates 5) Got samples out of freezer, pipetted designated vol. into centrifuge tubes as per my calculations; vortexed, centrifuged lightly. 6) Transferred mass spec-ready mixtures to autosampler vials; instead of pipetting 15ul I used 14.7, since using 15ul consistently resulted in air bubbles. Despite this, I had to re-pipette samples 54 & 55. 7) Added these newly prepared samples to the sequence table, continuing the pattern of QC, blank, 5 samples, repeat. 8) Loaded the samples on to Tray 2 in the following order:

1 2 3 4 5 6 7 8
B G041 G066 G105 G032 G129 G054 G081 G003
C G074 G014 G049 G053 G104 G055 G042 G064
D G073 G057 G007 G070 G001 G071-B G062
E GBlank OBlank

9) Added 65ul Final Solvent to the blank vial; there should be ~130ul blank as of 5:30pm today.

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Laura’s Notebook: Checking in on my oysters

Stopped by Manchester for the AM to check on things.

  • Downwellers inside were very dirty, holding lots of water but not overflowing. I cleaned them with fresh water.
  • I have 9 larval groups left (down from 16). Changed banjos. Water flow and food all looked good, no overflows.
  • Water change on SN growth experiment. This week I don’t have time to check these oysters out, but next week I plan to look at them under the scope, and assess whether or not I can count live/dead easily, and how I can keep them separate to potentially extend the experiment (is it even possible?)
  • Outside tank:
    • Drained tank
    • Rinsed downwellers and upwellers with fresh water. Downwellers were clean (since Olivia cleaned them on Monday), but upwellers were very dirty. After rinsing them, I uncovered some beautiful oysters! Here is the NF6 Ambient silo, which has a ton of juveniles: NF6 Ambient juveniles
  • I received the 1”->1/2” reducing couplers in the mail, so I set up one more downwelling string, and moved the HL-6 Ambient & HL-6 Low 180um setting silos outside.

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