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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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.

Wednesday 11/4/15 and Thursday 11/5/15

Wednesday 11/4/15

Gave a talk to the PSRF monthly meeting in the morning. Afterwards I went with Alice to the dock and brought up the cultch set F2 oysters to the hatchery. As there weren’t very many tile set oysters, I wanted to supplement the experiment with these. I put them in one of the setting tanks outside in static, ambient water.

Thursday 11/5/15 (Experiment Day)

Alice and I built a little “house” for the chiller to protect it from the rain. We set up the chiller in the smaller section of the setting tank in order to cool down the water quicker. The chiller was set to 0degC and turned on at 10am. Water on the other side was at 11degC, a little cooler than ambient due to the air temperature overnight. For the controls, 250+/- 5 cultch set oysters from each population were placed in 180 micron silos hanging in the ambient temperature side of the tank. I put around 350 oysters in silos on the chilled side at 11:30am when the temperature was at 7degC. At 1:00pm the temperature was at 0degC and maintained that temperature all day. At 1:30PM I added some tiles to the control and chilled tanks.

Chilled treatment on the left and controls on the right

Chilled treatment on the left and controls on the right

It's cold!

Tiles added to treatments. The numbers represent oysters on that tile after sampling for gene expression. F = front of tile, S = side, B= back
Control
20 hours 0degC
3 hours 0degC
HCA5: 12F, 1S, 13B
HCA13: 9F, 1S
HCA11: 10F, 3S
HCB2: 7F, 2S, 8B
HCA12: 8F, 1S
SSA9: 4F, 1S (4 sampled)
HCA2: 1F, 8B
HCB5: 1
NFA6: 2F (4 sampled)
SSA4: 4F, 2S
HCB8: 2B
SSA13: 4F, 1R
SSA1: 14F, 2S, 1B
SSB11: 1F
SSA11: 0 (4 sampled)
SSA14: 4F, 1S
NFA4: 14F, 1B
SSB2: 0F, 2B
NFA11: 6F, 1B
NFA7: 3F, 1S
NFA9: 4F
NFA12: 3F, 1B
NFB2: 10F
NFA15: 11F, 1B
NFA14: 5F
NFA2: 26F, 1B
NFA?: 15F, 1B
Total HC: 52
Total SS: 19
Total NF: 63
Total HC: 22
Total SS: 17
Total NF: 41
Total HC: 13
Total SS: 5
Total NF: 2

At 4:00pm I sorted out 200 cultch set oysters from the chilled tank and added them to new silos in the control tank labelled with the population name and “3 hr chilled”. In addition to looking for differential mortality, we are interested in looking at temperature-induced gene expression. I put some oysters from each group in small (300 mL) cups with chilled water and then dissected 6 from each population and put whole body tissue in 1.5 mL tubes with RNALater. This took about 45 minutes total but I tried to alternate between populations while dissecting. I took pictures of most dissected oysters with a ruler. At 5:00pm I brought in one tile for each population from the chilled tank and dissected 4 oysters per population, giving 10 samples each. These samples were placed in the fridge overnight. The other ~150 oysters in the chilled tank were left there overnight to be sampled the next day.

Time
10:00AM
– Chiller turned on and set to 0degC.
– Ambient at 11degC
11:30AM
– Chilled side: 7degC
– 350 cultch set oysters placed in chilled water
12:15PM
– Chilled side: 4degC
1:00PM
– Chilled side: 0degC
1:30PM
-Added tiles to control and chilled tanks
4:00PM
-Sorted out ~200 per population; added to new silo in the control tank
– Dissected 10 samples per pop for gene expression

Tuesday 11/3/15: Back in the Field!

On Sunday 11/1/15 I took an early flight out to Seattle for a week of torturing oysters. My goals for the week were to take pictures for size of my juvenile F2 oysters and do an experiment to test for differences in resilience to cold temperature shock.

Over the summer I set juvenile Olympia oysters on PVC tiles and hung them off the dock by the Manchester Research Station at the end of the summer. Unfortunately, one of my stacks of trays fell off the dock a few days after during a crazy storm. They sat on the bottom for a couple of days and then were rescued by a crew that left them sitting on the dock in the hot afternoon sun. Once the hatchery crew realized where they were they quickly put them in a tank, but the stack may have been out of water for a few hours. They were redeployed on 9/10/15 with safety line and haven’t had an issue since.

On 10/14/15 two of the PSRF hatchery crew, Stuart and Laura, pulled up the trays and photographed the tile to get size information. There was a lot of mortality on the stack that had been out of the water, but the other stack had good survival.

HCA11_10_14

Picture taken on 10/14/15 of a tile with Hood Canal juvenile oysters.

Today I pulled up the trays and took pictures of the tiles. It’s exciting to see how much they had grown, even in just 3 weeks!

Picture of Hood Canal oysters taken 11/3/15

Picture of Hood Canal oysters taken 11/3/15

I left the oyster tiles in a large, static tank at ambient temperature and splashed in some live algae. Alice and I also checked on some F2 oysters that were set on cultch at the end of the summer. These were basically leftover larvae after I had enough in my tanks with tiles. As larval production was really ramped down at the end of the summer, I suspect that these are from only a few individuals per population. To get a rough estimate of how many there were per population, I measured out 50 mL of oysters, counted them, and then measured the total volume of oysters.

  • Fidalgo Bay (NF): 215 oysters/50 mL in 450 mL total = 1935 total
  • Oyster Bay (SS): 254/50mL in 250mL total = 1270
  • Hood Canal (HC): 208/50 mL in 325mL total = 1352

At 9:30am we turned on a chiller in one of the outdoor setting tanks and set it to 0degC. I monitored it throughout the day to see how long it took to get to 0.

Time
Degrees C
9:30AM
13
11:00AM
8
12:00PM
6
1:15PM
3
2:30PM
0.5
3:00PM
0 (turned chiller off)

Monday 8/24/15 – Thursday 8/27/15 (Last week of fieldwork!)

Monday 8/24/15

Had a long chat with my advisor, Cathy, about the status of my fieldwork- what data I had collected, what I was leaving behind, plans for a stressor experiment- as well as plans for funding next summer. Puget Sound Restoration Fund has approved me to conduct a common garden experiment next summer with olys from California, Puget Sound, and British Columbia. My hope is to use this rangewide common garden to contextualize the regional-scale one I did this summer.

Tile culling

  • SSA_7: 9
  • SSA_8: 83
  • HCB_1: 160
  • HCB_2: 310
  • HCB_3: 384
  • Took pictures of tiles that were culled last Thursday but didn’t have pictures for.

Growth Rate Experiment

Started taking pictures of larvae from the growth rate experiments conducted earlier in the summer. This was done using an adaptor to attach a phone to the eyepiece of a microscope. Labelling scheme (until I think of a better one) is Population+Replicate_Growth rate experiment (1 or 2)_date sample was taken. Also did live/dead counts of the entire sample.

  • SS3_G1_7/12: 25L/15D
  • SS1_G1_7/12: 57L/8D
  • SS3_G1_7/15: 38L/14D
  • SS2_G1_7/12: 64 L/9D

Tile Set B

  • Cleaned out the tanks from tile set B. Cut off the tiles and randomized them among the wire “cages” in the large tank with the other tiles from Set_A.

Tuesday 8/25/15

Cultch Set

  • SS>450A + SS>450B -> SS> 450B; SS>1000
  • SS_new -> SS>450B; dump
  • HC > 450 ->  HC > 1000
  • HC_new -> HC > 450; dump

Tile culling

  • HC_B_1: 42
  • HC_B_2: 103 from front, 164 from back
  • HC_B_3: 67 front, 42 back
  • HC_B_4: 4
  • HC_B_7: 4 front, 13 back
  • HC_B_8: 12 front, 3 back
  • HC_B_10: 9 back
  • HC_B_11: 1 back
  • HC_B_12: 2 back
  • HC_B_14: 86 front, 5 back
  • SS_B_1: 7 back (none on front)
  • SS_B_2: 4 back (none on front)
  • SS_B_3: 0/3
  • SS_B_7: 0/10
  • SS_B_8: 0/2
  • SS_B_9: 0/5
  • SS_B_10: 0/7
  • SS_B_11: 0/6
  • SS_B_13: 0/1
  • NF_B_1: 0/27
  • NF_B_2: 95/120
  • NF_B_3: 0/13
  • NF_B_4: 77/270
  • NF_B_5: 8/12
  • NF_B_6: 208/92
  • NF_B_7: 0/2
  • NF_B_8: 18/10
  • NF_B_9: 8/19
  • NF_B_10: 70/48
  • NF_B_11: 11/17
  • NF_B_12: 10/27
  • NF_B_13: 1/12
  • NF_B_14: 0/2

Wednesday 8/26/15

  • Dropped off samples and borrowed materials at the Roberts lab.

Thursday 8/27/15

The little oyster babies are leaving the nest for the big open ocean! I started out the day finishing up any culling that was needed. I also looked over previously culled tiles to make sure that at least 1 cm of space was around each oyster.

Culling

20150911_121151

Steven Roberts came to help with the deployment. While I finished up culling, he made labels for the trays but cutting small PVC pipe into 1 inch pieces and etching numbers onto them. We’re also putting waterproof paper in a tube with the tray number.

12 tiles (4 per population) were attached to each tray with zipties. The populations were ordered in the same way for each section of each tray (NF, HC, SS).

Tray with tiles attached

Tray with tiles attached

Pictures were taken of each tile next to a ruler to measure size at deployment of the oysters.

HCA11_150827

7 trays were filled up with with tiles. To minimize the effects of location within a stack of trays, we put 4 trays in a stack with a 2′ spacer between the top 2 and bottom 2 trays. An additional tray was used as a cover to keep out predators. We made 2 of these stacks, with an empty tray in Stack 2.

Stack of trays with oyster tiles ready for deployment

Stack of trays with oyster tiles ready for deployment

Order of tiles and trays

20150911_12201820150911_122037

The trays were hung of the dock at Manchester by ~20 foot rope. One of the stacks seemed to float (which was odd), so we tied on clam bags with rocks to both stacks.

20150827_15371020150827_153657IMG_3008

Monday 8/3/15

Setting System

Last week, I decided to make a 2nd tank with tiles for each of the populations. This stemmed from not seeing as many setters as I’d like on the tiles in the 1st set-up, and because there were still so many in the “160” tanks. Also, having 2 tanks per population can mitigate “tank effects”- possible tank-specific differences that might confound population differences. I picked up some more PVC sheet, had it cut into 4″ x 4″ tiles at the store, and roughed them up in between today’s tank cleanings. To ensure enough larvae set per tile, I’m going to add up to 60,000 larvae per tank over the course of 1 week. While this means some larvae will be a few more days older than others, after an extended period of time this will be insignificant for measuring growth rate.

Did counts for NF_SetA and HC_SetA to get an idea of how many were left to set (ran out of time for SS_SetA).

Larvae tanks

Measured out the “160” tanks over 224, 200, and 100 to get an idea of how many 224s I will get over the next week.

  • NF_Tank2_160 (224) -> 13,406 total: 500 for DNA, 12,906 to NF_SetB
  • NF_Tank2_160 (200)
  • NF_Tank2_160 (100) -> swimmers only added back
  • NF_Tank1_new (100) -> swimmers only added back
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> 31,400 total: 500 for DNA, 30,900 added to HC_SetB
  • HC_Tank2_160 (200)
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> swimmers only
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 20,200 total, 500 to DNA, 19,700 added to SS_SetB
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> swimmers only
  • SS_Tank1_new (160) -> SS_Tank2_160

Tuesday 7/28/15 and Wednesday 7/29/15

Tuesday 7/28/15

New Larvae

  • HC: 0
  • SS: some
  • NF: 0

Settlement

Set up an “overflow” setting system for larvae that do not go into the tile set-up. Silos (15 cm diameter, 20 cm height) with 180 micron screens are suspended in a tote with a draining outlet. 224 sized larvae are added into the silos along with 1-3 tablespoons of 450 micron cultch. An airstone is in the totes and water/algae is dripped into the silos. The water level is 13 cm deep, making the volume of water in the silo to be 2,296 cm^3 (equalling to 2,296 mL). I’ve been told that ~5 larvae/mL is good for this type of set-up, with no more than 10 larvae/mL. At 7 larvae/mL, the max to add to each silo would be 16,000 larvae.

Wednesday 7/29/15

Larvae tanks

  • NF_Tank2_160 (224) -> 56,700 total: 600 for DNA, 15,300 to cultch set, 41,400 to NF_Tank2_160
  • NF_Tank2_160 (100) -> swimmers only added back
  • NF_Tank1_new (100) 0> swimmers only added back
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> 15,937 total: 600 for DNA, 15,300 added to cultch set
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> swimmers only
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 9,675 total, 600 to DNA, 9,075 to cultch set
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> swimmers only (a lot on bottom)
  • SS_Tank1_new (160) -> SS_Tank2_160

New larvae

  • Some new larvae from all 3 populations

Setting Systems

To clean the tile setting systems, I first fill up a clean, empty 100 L larval tank with seawater and fill up a misting sprayer designated for setters with fresh water. I empty a tile setting tank over a 100 micron screen to catch the larvae that haven’t set yet. I put these in a tripour beaker with seawater and place the tiles in the 100 L tank full of water. I clean the tank with Vortex and fill it up as quickly as possible. Meanwhile, I spray the tiles and poultry wire gently with freshwater before returning the tiles and larvae into the cleaned tank. This took a little getting used to, especially figuring out how to minimize the time the larvae sat in the beaker as they would try to set on the bottom if they were in there too long.

I initially thought that the cultch set would be an experiment to see if the populations have differential success in the number of single oysters produced. Because of this, I cared a lot about having replicate silos, adding similar numbers of larvae to each, adding larvae on the same day, adding the same amount of cultch to each, and randomizing the order of silos. This led to me not using all of the 224s that I screened out, particularly from NF which somehow peaked in the number of 224s before the other 2 groups. I’ve since realized that to truly make it a viable experiment would take a lot of work (especially in the number of silos needed once I started screening them out by size) and that the main benefit of the cultch set is to grow up F2 oysters for future experiments. So I regret throwing out some of the 224s, but should still have enough for the project goals.

  • NF: 15,300 added to Cultch_SetA
  • SS: 9,075 added to Cultch_SetA
  • HC: 15,300 added to Cultch_SetB

Wednesday 6-24-15

Steven Roberts came to help out today, which made the Wednesday cleaning/collecting larvae only take about 2.5 hours. He did all of the larval counts, filtered out the SS larval catches, and cleaned many of the buckets. We decided in the interest of time to just qualitatively note individual family output of larvae and then combine the larval output from each family in a population for counting.

Families that were filtered for larvae:

  • very little/some larvae: HC2, SS5, SS2, SS4, SS3
  • lots: NF5, HC3
    • for both of these the larvae seemed to be clustering near the bottom (dead??)
SS5 seemed to release some sperm when put in the bucket with all the other SS families.
I’ve organized my datasheet for larval counts a little differently.
  • The 1st sheet, “Day 1”, is the raw data from counting the larval output from the broodstock.
  • The 2nd sheet, “Larval tank counts”, is the raw data from counting the number of live/dead larvae filtered out of the 100 L larval tanks. It also lists the estimated ages of the cohorts in the tanks.
  • The 3rd sheet- “Larval tanks running total”- is formatted like a bank account transaction list, with larval tanks for each population the “accounts”. This incorporates data from the first 2 sheets to let me know approximately how many larvae I have in the 100 L tanks at a time. The differences between counts is also my estimated mortality (although some of this is due to leakage during cleaning).

Update 6-25-15

  • On Tuesday 6-23-15, the seawater was shut off for about 3 hours. Also, there was an issue with the airline so that tanks may not have been bubbling for a while. When I went in on Wednesday, all tanks seemed to be bubbling fine.

Setting up an Oyster Garden

Monday (June 8) was my first day out at the NOAA Manchester Research Station in Washington State. Specifically, I’m working in the Kenneth K. Chew Center for Shellfish Research and Restoration. This shellfish hatchery is the result of collaboration between many groups and funding agencies, in particular the Puget Sound Restoration Fund (PSRF).

http://www.nwfsc.noaa.gov/news/features/hatchery/

The hatchery (right) and algae greenhouse (left)

My project this summer is to raise oysters descended from three Puget Sound populations under common conditions in order to measure differences in fitness. This type of experimental design is commonly referred to as a “common garden”, and allows one to control environmental variables so that phenotypic disparities among individuals can be attributed to their genetic differences. My fitness metrics are reproductive output, survivorship at different life stages, and growth rate. I will also be taking DNA/RNA samples along the way to see if mortality is random in respect to genotype, or due to purifying selection. With the RNA, I plan to look at differences in gene expression to help detect cryptic differences in phenotype between these populations.

Three source populations for common garden experiment

Three source populations for common garden experiment

This project is a collaboration with Steven Robert’s lab at the University of Washington, who previously conducted a reciprocal transplant experiment with offspring of wild oysters from these same populations. For that experiment, they outplanted the young oysters from each group at four different sites and measured growth rate, mortality, and reproductive characteristics. They observed significant variation at these metrics among populations and sites (informative slides and manuscript preprint available here). My experiment will be following up on these results by testing if population-level differences are consistent in a second generation under controlled environmental conditions.

As I’ve never raised shellfish before, this week has had a bit of a learning curve. Fortunately for me, the staff at the hatchery have been super helpful in showing me the ropes and advising on how to set up my experiment. I’m starting with about 100 adult oysters for each group (see lab notebook entries for data). These are the first generation (F1) offspring of wild oysters, and have been living in common conditions their entire lives- mostly hanging off the docks near the hatchery. Their offspring will be 2nd generation (F2) from the original broodstock, and should have any influence from maternal effects erased.

The adults were brought in to the hatchery on May 28 and placed in three separate buckets to avoid cross fertilization. To maximize genetic diversity and minimize the chance that one male fertilizes all of the females, I split each group into 5 buckets of ~20 oysters. These “families” will be marked, so that I can genotype them later and follow their offspring’s success throughout the experiment. Their water temperature was switched to a balmy 20°C this week, which will encourage them to start spawning and producing larvae.

(sorry for the lack of pictures, I’ll take some and put them up soon!)