Day 0 Sampling- Olympia Oyster OA Project

For my last morning in Washington state, Sam White and myself dissected 48 Olympia oysters for 3 different tissue types as part of an adult Olympia oyster/rock scallop ocean acidification experiment. Sam already wrote up a great summary of the day in his lab notebook. A list of all adult oysters sampled this summer, including those used in the adult OA experiment, can be found here or at the appropriate link under “Datasheets” in this lab notebook. This datasheet includes the date dissected, treatment tub, weight in grams, reproductive status at time of sampling, date of mortality (if oyster died prior to being dissected), and which tissues were samples. To measure size of oyster, I have labelled pictures of every oyster with a ruler in a Dropbox folder. I also wrote up a dissection protocol for subsequent sampling days.

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Setting up for adult Olympia oyster OA experiment (20160907-11)

Wednesday 2016-09-07

In the morning, Natalie and I went to the Taylor Shellfish hatchery to pick up valves for the broodstock OA system. We got to Manchester around 12:30pm and worked on finishing up the plumbing and gluing the PVC. The hoses that were intended to be used to deliver water to the tubs didn’t fit easily on the valves, so we stuck one end in a toaster oven for a minute to make it easier to push on. We hooked up the manifolds to the OA system and let water run through overnight. Then we realized we hadn’t added in coupling for the algae injection port (D’oh!), so that needed to be addressed on Thursday.  We didn’t have time to give algae to the oysters or scallops, so both animals ended up without food overnight.

Oyster Mortalities

  • CA1T (1), BC1T (1), BC2T_058

Need to do: get brand name of valves

Thursday 2016-09-08

  • Used the rest of the white shellfish tags to label oysters that will be used in the adult OA experiment
    • Weighed oysters before adding tag and recorded their tray group during the larval experiment
    • Placed them in a single layer on the bottom of flow-through buckets with seawater and live algae overnight for putty to cure

20160909_132205

  • Checked for mortalities
    • 2 from OR1T (sampled 1)
    • 1 from OR2T (sampled)
  • Figured out the flow and food concentration with Ryan for the scallop tanks
    • Goal is to have at least 100,000 cells per mL of algae
    • Found that when valves are set to 50 the flow into each tub was ~0.85 L/min
    • Added couplings for the algae injection port on the flex hoses leading up to the manifolds
  • Starting around 5pm, took the scallops out of their fish totes and divided them up evenly amongst the 8 treatment tubs, with 11 or 12 scallops per tub. There were 4 fish totes corresponding to the 4 source populations.
    • Scuzz and some soft tissue epiphytes were gently wiped off with gloved hands. Scallops placed so they would not be touching each other
    • Temp of tubs: 15.12degC – 15.37degC, Salinity: 28.6
    • Tote A2 scallops spawned
      • Looked like eggs from 380, 311, 48, 381
    • Tote B2 spawned
      • just eggs from 312
Tub 1A Tub 1B
  • Tote 5: 305, 302
  • Tote 4: 363, 360, 244
  • Tote 3: 63, 45, 241, 57
  • Tote 1: 375, 391, 390
  • Tote 5: 310, 303
  • Tote 4: 240, 361, 358
  • Tote 3: 66, 36, 65
  • Tote 1: 373, 372, 379, 383
Tub 2A Tub 2B
  • Tote 5: 311
  • Tote 4: 246, 359, 237
  • Tote 3: 48, 53, 58, 64, 67
  • Tote 1: 392, 381, 380
  • Tote 5: 312
  • Tote 4: 236, 242, 248
  • Tote 3: 51, 53, 39, 56
  • Tote 1: 374, 370, 387
Tub 3A Tub 3B
  • Tote 5: 309, 300
  • Tote 4: 362, 234, 245
  • Tote 3: 44, 38, 43
  • Tote 1: 377, 388, 393, 384
  • Tote 5: 308, 301
  • Tote 4: 364, 233, 231
  • Tote 3: 37, 50, 54
  • Tote 1: 389, 386, 376
Tub 4A Tub 4B
  • Tote 5: 306, 304
  • Tote 4: 237, 239
  • Tote 3: 59, 52, 60
  • Tote 1: 371, 395, 385, 46
  • Tote 5: 307
  • Tote 4: 243, 247, 238
  • Tote 3: 41, 61, 62, 47
  • Tote 1: 382, 378, 394

 

Friday 2016-09-09

  • Checked for mortalities
    • BC 049
  • Divided up oysters and placed them into clam bags to hang in treatment tubs with scallops.
    • 12 British Columbia oysters per treatment tub
    • 10 California and 10 Oregon oysters per treatment tub
  • Dissected adductor muscle from extra BC oysters not used in OA experiment
    • Recorded weight, reproductive status, tray group, and took pics for size
    • BC1T A, B, 93, C, D, 101
    • BC2T A, 111, 110, B, C, D, E, 108
  • Checked algae in one A tub and 1 B tub using Coulter counter
    • 92,921 cells/ml, 99,070 cells/ml

 

Sunday 2016-09-11

  • Recorded which oysters were in which experiment tub and made sure numbers were balanced, in case an oyster gets misplaced when cleaning
  • Checked for mortalities
    • 024W (CA)
    • 058W
  • Weighed and took pictures for size of any oysters I did not already have data for
  • Dissected adductor muscle into RNALater from 12 oysters that were in tray OR1T during larval experiment
    • Labelled OR1T A-L
  • Processed the rest of the tiles from the acidification larval experiment
    • These are 4.5in diameter PVC tiles that were roughed up on both sides with sandpaper and placed with oyster larvae 14 days post-release
      • Only added if at least 20% survival in treatment
    • To process them I counted the number of live and dead oysters per tile, took a pic of both sides with a ruler, and scraped off juveniles into RNALater if there were at least 5 living oysters on the tile

2016-09-01

Thursday 2016-09-01

  • Checked for mortalities
    • CA1T (2), CA4B (2), CA2T (1), 011 from OR1T
  • I bought 150 white shellfish tags (1/8″ x 1/4″) labelled 000-149 in order to label some of the oysters to be used in the OA experiment. This is to help determine which population an oyster belongs to in case it gets mixed up during sampling.
    • Sean used seawater epoxy to attach tags to some of the oysters that will be used in the adult OA experiment. Before attaching tags, he recorded weight for the oyster in grams. He tried to randomize the oysters chosen for the experiment by size.
    • Since some oysters already had orange shellfish tags (labelled 001-074) from the summer larval experiment, all subsequent labels will have a “W” to denote the ID of an oyster with a white tag. To cure the epoxy, these oysters were left in a single layer on the bottom of a bucket with an airstone and flow-through of algae and seawater.

epoxy

  • I took pictures and counted the number of live oysters on some of the tiles from the larval experiment, as well as emptied out some silos that still had larvae in them. If any larvae had set on the side of the silos, I counted them and scraped them off into a 1.5 mL tube with RNALater. Tiles with oysters on them were left hanging in a bucket fed by the oyster broodstock manifold.
    • Tiles w/oysters: B61, B89, B67, A48
    • Tiles w/out oysters: A82, A71
    • Silos: A26, A48, A27
  • I dissected adductor muscle from some California and Oregon oysters that were not going to be used in the adult OA experiment.
    • CA4B_A, CA4B_B, CA4B_C (tiny oyster attached to CA4B_B),CA4B_D, CA4B_E, CA4B_F, CA4B_G, CA4B_H, CA4B_I, CA4B_J, CA4B_K
    • OR2T_E, OR2T_F, OR2T_G, OR2T_H, OR2T_I, OR2T_J, OR2T_K, OR2T_L, OR2T_M, OR2T_N, OR2T_O, OR2T_P, OR2T_Q, OR2T_R
    • OR5B_A, OR5B_B
    • OR2B_A, OR2B_B (attached to C), OR2B_C (attached to B), OR2B_D, OR2B_E, OR2B_F

 

Wed. 2016-08-31

Wednesday 2016-08-31

  • Screened out silos from the new new experiment started ??. Day 7
ID Family Sample Action
B24 EtOH Dumped
A16 EtOH Dumped
B37 250 in RNALater, 100 in EtOH Larvae returned to clean silo
  • Counted and took pics of tiles that had already been removed from silos. For tiles that had at least 20 oysters I sampled ~10 oysters and stored in RNALater.
ID Family Sample Action
A46 No Had oysters, replaced
A10 11 Had oysters, replaced
A90 10 Had oysters, replaced
A85 13 Had oysters, replaced
B58 16 Had oysters, replaced
B64 all oysters Not returned, no pic
B54 10 Had oysters, replaced
A84 0 No oysters, not returned

Monday 2016-08-29

Monday 2016-08-29

  • Screened out silos from new new experiment started ?? for Day 7
ID Family Sample Action
B32 EtOH Dumped
A17 EtOH Dumped
B70 EtOH Dumped
A15 500 in RNALater, 100 in EtOH Larvae returned to clean silo
  • Counted newly released larvae
    • over 10,000: CA2_T, OR5_B, CA4_B (mostly dead)
  • Screen out silos with tiles in them. Counted planktonic larvae in silo, spat on sides of silo, and spat on tiles. Rinsed off tiles gently with freshwater.
ID Family Sample Action
A27 No Replaced tile and larvae in clean silo
A45 No Replaced tile and larvae in clean silo
A48 No Replaced tile and larvae in clean silo
A46 EtOH Tile returned to tank, larvae dumped
A83 EtOH Culled some from tile, tile returned to tank, larvae dumped
A82 EtOH Tile returned to tank, larvae dumped
A23 EtOH Tile empty, not returned to tank
A49 EtOH No count for tile, not returned to tank

Start of full-scale larval OA experiment!

Wednesday, 2016-07-27

Today started like many of the previous days, with checking for extruded eggs and counting the larvae in some of the silos from the preliminary experiments. I had a late start at the hatchery because I was picking up a pipette gun from campus.

Extruded Eggs:

  • BC3T
    • 2 new: 112(2), 113(2)
    • 1 repeat: 34 (1)

Screened out silos at Day 5 from experiment started 2016-07-22 with OR2T and CA2T larvae. Took pictures under microscope at 10x of all but A84 and A83.

ID Family Sample Action
A86  OR2T 750 in RNALater Put in clean silo
A85  OR2T 750 in RNALater Put in clean silo
B61  CA2T 750 in RNALater Put in clean silo
B59  CA2T 750 in RNALater Put in clean silo
B65  OR2T 750 in RNALater Put in clean silo
B63 OR2T 750 in RNALater Put in clean silo
A84  CA2T 750 in RNALater Put in clean silo
A83  CA2T 750 in RNALater Put in clean silo

Then Sean and I started screening out the newly released larvae from the trays and buckets. There did not seem to be enough larvae from all three populations, so I stayed to finish screening out the last couple of buckets and count larvae. While the BC3T oysters were sitting in a bucket waiting to be placed into a clean tray, they started to release A LOT of larvae. Once I finished screening the last of the buckets I realized that we finally had enough from all three populations to start the experiment. Hooray! However, it meant I ended up staying until 10pm setting up by myself.

I had to remove some of the silos from preliminary experiments that were in the system in order to make room for the full experiment. I screened these out and left in tripours overnight to count the next morning.

  • B72, A63, A6, B66, B60, B55, A88, B66, B80, A100, A97, B51, A9

There were two family groups for CA and OR that had a lot of larvae, whereas BC had one group with a lot of larvae and one group with ~20,000. I decided to combine the BC groups, then have 2 replicates per treatment for each family group, so 4 silos per population/ treatment for CA and OR and 2 silos per population/treatment for BC. Not ideal, but I wasn’t sure when I would have at all three populations spawning at the same time again. I counted the larvae for each population with 4 subsamples of 0.5 mL, took the average to get larvae/mL and then calculated how many mL to add to each silo to get 15,000 larvae. I dispensed larvae with a pipette gun and 25mL pipettes while plunging constantly to mix, using a new pipette for each family group. When pipetting out replicates, I alternated between treatments to help mitigate possible effects of the plunging/pipetting process (probably a little overkill).

Family larvae/mL  mL added Experiment labels
OR1T  152.8 98.2 B20, B18, A72, A73
OR5B  153 98 A59, A71, B13, B11
CA4B  168 89.3 B19, A58, A57, B06
CA2T  290 51.7 A55, A56, B16, B12
BC3T+BC2T  232.5 64.5 A53, A54, B35, B45

I had hoped there would be enough larvae from BC1T, but there was only ~10,000.

Broodstock Setup

Out with the old…

In a classic fieldwork moment, I had just finished setting up one system for my broodstock, when a newly published paper and a phone chat changed my mind and I had to almost completely redo it. Initially, I had decided to split my adults into 4 or 5 buckets per population of ~25 oysters each in order to maximize genetic diversity by minimizing the chance of one male fertilizing all females. I built a manifold for this system (new term I learned meaning “pipe branching into several openings”) and had 0.5 gal/hour drippers delivering 16degC seawater and algae to the buckets. The broodstock were taken out of their combined tank on Tues. May 31 and separated into a single 5 gal bucket per population. They were split up into ~25 per bucket on Wed. June 15, with 4 buckets for California and 5 buckets each for BC and OR. My cleaning schedule for buckets was to rinse them and wash with Vortexx 3 times a week, and change out the drippers every day I was at the hatchery with ones that had been sitting overnight in Vortexx.

Then on Friday June 17 I read this new paper out of George Waldbusser’s lab at OSU. They conducted a carbonate chemistry manipulation experiment with fertilized eggs extracted from brooding Olympia oysters. A quick reminder- male Olympia oysters release sperm into the water column which is then filtered out by females for fertilization. The females then keep the fertilized eggs in their mantle cavity for up to 12 days before releasing the now weakly swimming larvae. In Waldbusser et al. 2016,they found that larvae extracted ~36 hours after fertilization developed at a similar rate as larvae left in the brood chamber. As far as I know, this was one of the first experiments where Ostrea larvae had been raised outside of the brood. In order to identify a newly fertilized female, they looked for eggs that had been essentially spit out by an adult oyster. Chatting with Matthew Gray from the project was really helpful in figuring out how I can adapt my system to identify brooding females and possibly try to run some experiments of my own on newly fertilized larvae.

…in with the new

On Thursday June 23, I did away with most of the buckets and laid out oysters 2″ apart in black plastic 9 gal tubs (originally meant for cement mixing), with 40-50 oysters per tub and 3 tubs for BC and 2 tubs for CA and OR making 7 tubs total. This number was primarily dictated by space, or else I would’ve split them up into more trays. Some oysters were stuck together in a way that made it impossible to separate without killing them, and so were put in 3gal buckets with small 100 um banjo filters. Each tray has an airstone, either two 1/2 gal/hour drippers or a single 1 gal/hour dripper, and a banjo filter.

Cleaning these trays is a pain as they are too heavy and awkward to pick up and pour out into a bucket, so we use a hose to drain the tray into a bucket and keep the broodstock in a clam bag and separate bucket of seawater during cleaning and filtering for larvae. As we screened out the first batch of larvae on Thursday, we will be cleaning all trays/buckets every day to check for larvae. The official start of the experiment will be when all three populations are releasing enough larvae.

Back in the swing of things

Friday May 13 was my first day back out at Manchester. This summer I will be conducting an acidification stress experiment on Olympia oyster larvae from San Francisco Bay, CA, Coos Bay, OR, and Ladysmith Harbor, BC. Wild adults were collected from each site in December and January and shipped in coolers to a quarantine room at the Manchester Research Station so that the parents may experience the same over-wintering conditions together. They were kept in the same large tank in separate, triply labelled clam bags and fed a mix of live algae from the hatchery and algae paste.

Since arriving in Washington, I’ve mostly been getting my experiment system ready and conditioning my broodstock for spawning. There have been some logistical issues, as a rock scallop project also housed in the quarantine room hasn’t completed yet so we’ve had to adapt the OA system for the possibility of both experiments running at the same time. With many Home Depot/Harrington’s Plastics runs and hours of fitting PVC pipe together, it has felt like slow going. At the very least, if science doesn’t work out I think I’m now qualified to be an assistant plumber!

Attached below is a todo list document I made at one point to help keep track of all the things that needed to be accomplished.

Exp_5_27_16

One great improvement for this summer over last is the help of a part-time intern, Sean, who recently graduated from the University of Washington. As someone who has extensive experience building and running shellfish experiments through his work with the Friedman lab, I’m sure I couldn’t complete this project without his help.

 

Wet Lab posts on Benchling

A few weeks ago I made 2 more Genotype-by-Sequencing libraries for my Olympia Oyster population structure study, made up mostly of redos and some samples from the sister species Ostrea conchaphila. I tried out the free version of Benchling for it and really liked the interface and how it organizes protocols with your notes. I’ll continue using it for my wet lab notes and post the links to my entries here. All field work posts and analysis updates will still be posted here. The link to my Benchling is:

https://benchling.com/ksil91/

This link is on my About page as well.