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.

Advertisements

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

Wednesday 8/12/15- Friday 8/14/15

Wednesday 8/12/15

Larvae tanks

  • NF_Tank2_160 (224) -> NF cultch set
  • NF_Tank2_160 (100) -> swimmers only
  • HC_Tank2_160 (224) -> HC cultch set
  • HC_Tank2_160 (100) -> swimmers only
  • SS_Tank2_160 (224) -> barely any, put back in tank
  • SS_Tank2_160 (100) -> swimmers only

Tile Set

  • Did live/dead counts for both A and B
  • Did not add the unset larvae back to A
  • Rinsed tiles

Cultch Set

  • NF >450 -> NF >1000
  • SS > 450 -> SS>1000
  • SS_SetA -> SS > 450; SS_SetA

Thursday 8/13/15

Started dissections of tissue from the broodstock for subsequent DNA extractions with the help of Brent Vadopalas and the PSRF intern Ryann. First, we recorded the weight of each oyster in a family and placed it on a numbered pad.

Broodstock dissection setup

Broodstock dissection setup

A picture was taken of the entire family with a ruler for later measurement in ImageJ. Effort was made to hold the phone level to avoid the impact of tilt on apparent size. We made a little assembly line, with Brent shucking before joining Ryann and I in dissecting out adductor muscle tissue and storing in 1.5 mL tubes with 1 – .75 mL RNALater. If there was not very much muscle tissue, mantle or the entire oyster were taken as well. Scalpels and forceps were rinsed sequentially in soapy water, bleach, and freshwater between each oyster. Fresh scalpels were used between populations. We got through SS2, SS1,SS3, SS4, SS5, and NF4, averaging about 45 minutes per family by the time we got the hang of it.

Husbandry

  • Rinsed tiles and cultch
  • Feeding

Friday 8/14/15

Larvae tanks

  • NF_Tank2_160 (224) -> NF_New cultch set
  • NF_Tank2_160 (100) -> dumped
  • HC_Tank2_160 (224) -> none
  • HC_Tank2_160 (100) -> dumped
  • SS_Tank2_160 (all) -> dumped

Tile Set Counts

  • NF_SetA
  • SS_SetA
  • HC_SetA
  • HC_SetB
  • SS_SetB

Dissections

Did NF2 and NF1 with a little help from Ryann on NF1