Laura’s Notebook: Acute OA Exposure on 2017 Oly seed

June 1st, 2018

Ran a short term acute low pH stress trial on a subset of my Fidalgo Bay and Oyster Bay seed (Olympia oyster) that I grew in 2017. The trial was conducted at the Point Whitney shellfish hatchery in the OA system that Steven, Brent and Sam developed for geoduck trials this summer.

The acute OA exposure was 7.0 pH, with control at pH 8.16. I selected 2 Oly subpopulations from my 2017 seed: Fidalgo Bay (NF), and my South Sound (SN, from Oyster Bay) oysters from the 2017 mini-silo experiment. NF was selected because they showed a phenotypic difference (lower survival) associated with parental low pH exposure. SS was selected because they did not; the mini-silo animals from the SS population were selected because they likely have the least degree of genetic difference within treatments, as they were pulled from a single larval release date.

Sample key and water quality data are currently housed in this Acute-OA-Trial.xlsx Excel file

Schedule of events

Picked up oysters from Manchester, 8 groups in total, and transferred to Point Whitney hatchery:

  • NF 6C – Ambient pH
  • NF 6C – Low pH
  • NF 10C – Ambient pH
  • NF 10C – Low pH
  • SN 6C – Ambient pH (Mini-exp)
  • SN 6C – Low pH (Mini-exp)
  • SN 10C – Ambient pH (Mini-exp)
  • SN 10C – Low pH (Mini-exp)

Selected 24 of the largest but (approximately) equal sized oysters from each group – this was somewhat difficult due to different sizes between groups, randomly divided into 2 groups of 12 and sealed into fiberglass pouches made from window screen. Mean and size ranges are as follows:

Mean Min length Max length
All animals 14.1 7.7 19.1
NF 6 Amb – pH Treatment 13.6 12.2 15.5
NF 6 Amb – Control 13.7 11.4 17.0
NF 6 Low – pH Treatment 11.8 10.0 15.0
NF 6 Low – Control 11.0 8.9 15.7
NF 10 Amb – pH Treatment 15.5 11.4 17.9
NF 10 Amb – Control 14.7 12.4 16.1
NF 10 Low -pH Treatment 15.2 11.9 18.2
NF 10 Low – Control 14.3 10.9 17.2
SN 6 Amb – pH Treatment 14.7 11.8 18.5
SN 6 Amb – Control 14.6 13.1 17.1
SN 6 Low – pH Treatment 15.7 13.2 18.4
SN 6 Low – Control 14.5 12.0 19.1
SN 10 Amb – pH Treatment 17.8 16.9 18.8
SN 10 Amb – Control 17.6 16.2 19.1
SN 10 Low -pH Treatment 15.2 11.9 18.2
SN 10 Low – Control 13.3 7.7 16.6

Low pH Treatment

Two conical flow-through (rate?) tanks were equiped with a CO2 bubber connected to an APEX system, which continuously read pH, and injected CO2 if pH was above the set point. Set point was 7.0, and during the treatment pH held very closely to this – between 6.98 – 7.02. pH was recorded with the APEX system, but unfortunately it’s not clear how to extract this continous data … will check with Sam to see if he figured it out, he was working on this.

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Water samples

At time 0, 2hr and 4hr water samples were collected for total alkalinity. 125mL glass bottles were submerged inverted, filled, injected with 50uL mercuric chloride, capped and mixed by inverting several times. At the same time other water quality parameters were recorded (pH, temperature, salinity) using the APEX system, and separately a benchtop Accument pH/temp probe in 2L grab samples from each tank, and refractometer for salinity. I also had Rick’s Sond, but its batteries failed just before hour 2, so I wasn’t able to use that for subsequent measurements.

Summary of water quality measurements:

Time Sensor Tank pH Temperature Salinity Sample Type TA Sample Label (2 reps per tank)
13:00 APEX pH Treatment 7 16.2 23.1 In Tank Time = 0
13:00 APEX Control 8.13 16.4 23.1 In Tank Time = 0
13:30 Accumet Standard – 4 4.107 19 NA Standard NA
13:30 Accumet Standard – 7 7.063 19.1 NA Standard NA
13:30 Accumet Standard – 10 9.628 19.1 NA Standard NA
13:45 APEX pH Treatment 6.98 16.2 23.1 In Tank NA
13:45 APEX Control 8.16 16.5 23.1 In Tank NA
13:45 Accumet / Refractometer pH Treatment 7.015 14.6 26 2L grab sample NA
13:45 Accumet / Refractometer Control 8.297 14.8 26 2L grab sample NA
13:45 Sond pH Treatment 7.49 14.83 27.8 In Tank NA
13:45 Sond Control 8.35 15.04 27.8 In Tank NA
14:45 APEX pH Treatment 7.01 16.2 23.1 In Tank TIME = 2hr
14:45 APEX Control 8.16 16.5 23.1 In Tank TIME = 2hr
14:45 Accumet / Refractometer pH Treatment 7.06 14.6 26 2L grab sample TIME = 2hr
14:45 Accumet / Refractometer Control 8.205 14.8 26 2L grab sample TIME = 2hr
14:45 Mercury Thermometer pH Treatment NA 14 NA In Tank TIME = 2hr
14:45 Mercury Thermometer Control NA 14 NA In Tank TIME = 2hr
15:45 APEX pH Treatment 7.01 16.2 23.4 In Tank NA
15:45 APEX Control 8.17 16.5 23.4 In Tank NA
15:45 Accumet / Refractometer pH Treatment 7.032 14.6 26 2L grab sample NA
15:45 Accumet / Refractometer Control 8.214 14.7 26 2L grab sample NA
16:45 APEX pH Treatment 7.01 16.2 23.6 In Tank TIME = 4hr
16:45 APEX Control 8.17 16.5 23.6 In Tank TIME = 4hr
16:45 Accumet / Refractometer pH Treatment 7.019 14.5 26 2L grab sample TIME = 4hr
16:45 Accumet / Refractometer Control 8.21 14.8 26 2L grab sample TIME = 4hr

Tissue sampling

At 5.5 hours I began sampling whole tissue from the oysters. Since I had 172 animals to dissect I had to make some decisions about sampling scheme. Concerns included:

  • Time out of treatment before sampling – the longer the time, the greater the proteins related to anaerobic metabolism would be present
  • Time in the pH treatment

Since I plan to initially only sequence the NF population, only 6C group to start, I prioritized sampling animals from each group all at once to minimize the time out of treatment before frozen. Thus, I removed treatment/control groups simultaneously from the tanks, then sampled. It took ~20 minutes to sample 24 animals. Sampling method for RNA/protein is tricky, particularly if you’re sampling a ton of animals. Something I’d like to discuss with the lab group for future projects, particularly for the Oly eelgrass outplant!

Sampling protocol: Measured oyster maximum length (from hinge) using digital calipers. Used scalpel to shuck oyster, scraped all tissue from shell into 1.7mL microcentrifuge tube, froze in liquid nitrogen and held on dry ice. Between animals scalpel was wiped clean, rinsed with 10% bleach + fresh water solution, then rinsed well with fresh water. I did not have DI water (whoops, forgot to bring – how important is that? Should ask lab.), so I used fresh water from the hatchery hose. Sample were transported back to UW that night and put into the -80 at ~midnight.

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The view as I left the hatchery was spectacular

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