I drafted a narrative summary of the Olympia oyster experiment conducted over the past year to orient visitors to my GitHub repo and to the project. Check it out. (by the way, it’s much prettier to view in GitHub than on my webpage)

Climate fitness and adaptation in Ostrea lurida via transgenerational inheritance


Populations

In December 2016 four groups of Ostrea lurida were moved from pearl nets hanging off the NOAA dock in Clam Bay, Manchester WA into the the Ken Chew Center’s hatcery. Populations include:

  • Three groups of first-generation hatchery-produced (F1) oysters, all hatched at Port Gamble in 2013 by Puget Sound Restoration Fund staff and Jake Heare, UW SAFS graduate student. The broodstock used to produce these F1 oysters were wild, harvested from Fidalgo Bay in North Puget Sound (NF, for “North-Fidalgo”), Dabob Bay in Hood Canal (HC, for “Hood Canal”), and Oyster Bay in South Puget Sound (SS, for “South South”).
  • One group of second-generation hatchery-produced (F2) oysters (K, for “Katherine”), hatched at the Ken Chew Center in 2015 by Katherine Silliman, University of Chicago graduate student. The broodstock used to produce these F2 oysters were the aforementioned F1 oysters from Oyser Bay in South Sound.

Figure: Sites where Oly populations’ progenitors were collected (F, D, O) and where Olys were housed prior to experiment (C). Source: Heare JE, White SJ, Vadopalas B, Roberts SB. (2017) Differential response to stress in Ostrea lurida as measured by gene expression. PeerJ Preprints 5:e1595v3
Oly population sites


Temperature Treatment

Each of the 4 Oly populations were divided into 2 treatments and “overwintered” From December 6th to February 4th in two temperatures: 6degC, 10degC which represents historic and projected winter temperatures, respectively. While each population was divided into two bags per treatment (4 bags total per population), two overwintering tanks were used, one per temperature.

Table 1: Number of oysters during the temperature treatments for each population, with -1 and -2 denoting “replicates”:

  6 degC 10 degC
K-1 168 170
K-2 148 167
SS-1 52 51
SS-2 50 48
HC-1 28 29
HC-2 27 27
NF-1 46 51
NF-2 53 47

Temperature treatments were terminated on February 4th. A subset of animals from each treatment from SN, HC & NF animals were sampled on February 4th, and K on February 11th, for histology and tissues. Check out Steven’s video for a time-lapse of the sampling, and my [Lab notebook posts from 2/4 & 2/11 for images and details. Breifly, all sampled animals were weighed (g), measured (mm), shucked, and tissue was isolated. Tissue samples included: mantle (M), ctenidia (C) & adductor (A) into 2mL centrifuge snap-top tubes, immediately flash-frozen in an ethanol/dry ice bath, and kept on dry ice until transported to the -80 in Rick’s lab. The remaining visceral mass sampled for gonad was put into histology cassettes. Any residual tissue (gill, mantle or adductor) was sampled for DNA by placing into tubes with 1mL ethanol, and stored at room temperature.

Table 2: Number of each population sampled post-temperature treatment; see 2017-02-04_SamplingData.xlsx for details.

  6 degC 10 degC Date Sampled
K 15 15 2/11/2017
SS 15 15 2/04/2017
HC 9 9 2/04/2017
NF 15 15 2/04/2017

pH Treatment

On February 15th oyster groups were again divided in half for a 2-month pH treatment, at pH ~7.8 and ~7.2 constituting ambient and low pH, respectively. Animals were housed in six 50L flow-through tanks, 3 replicates per pH treatment, were fed continuously with Reeds shellfish Diet via dosing pump, and co-habiatated with ~20 large Crassostrea gigas per tank. Temperature was measured continuously via HOBO Data Loggers and discrete water samples were collected 3x/week with simultaneous T, S & pH parameters measured.

pH treatments were terminated on April 8, and a subset of SN, HC, NF were again sampled on 2/8 and K on 2/13 using the same protocol as the February sampling dates. All samples were again labeled and housed in Rick’s -80 freezer.

Table 3: Number of each population sampled post-pH treatment; see 20170408_Sampling-Data.xlsx

       
Sample Label Population Treatment # Sampled
SN-6-16 to SN-6-24 SN chilled T, low pH 9 (3/rep)
SN-6-26 to SN-6-33 SN chilled T, ambient pH 9 (3/rep)
SN-10-16 to SN-10-24 SN ambient T, low pH 9 (3/rep)
SN-10-25 to SN-10-33 SN ambient T, ambient pH 9 (3/rep)
NF-6-16 to NF-6-24 NF chilled T, low pH 9 (3/rep)
NF-6-25 to NF-6-33 NF chilled T, ambient pH 9 (3/rep)
NF-10-16 to NF-10-24 NF ambient T, low pH 9 (3/rep)
NF-10-25 to NF-10-33 NF ambient T, ambient pH 9 (3/rep)
HL-6-10 to HL-6-15 HL chilled T, low pH 6 (2/rep)
HL-6-16 to HL-6-21 HL chilled T, ambient pH 6 (2/rep)
HL-10-10 to HL-10-15 HL ambient T, low pH 6 (2/rep)
HL-10-15 to HL-10-21 HL ambient T, ambient pH 6 (2/rep)
K-6-16 to K-6-30 K chilled T, low pH 15 (5/rep)
K-6-31 to K-6-45 K chilled T, ambient pH 15 (5/rep)
K-10-16 to K-10-30 K ambient T, low pH 15 (5/rep)
K-10-31 to K-10-45 K ambient T, ambient pH 15 (5/rep)

Conditioning for Spawn

On April 11th remaining Olys were moved into a single flow-through tank equipped with a recirculating heater to condition them for spawn and house them while I replumbed/reconfigured the space. Temperature was raised ~1degC/day to reach the target temperature of 18degC, however the heater was not capable of maintaining 18degC, so the actual temperature in this setup was closer to ~15degC. Olys spent ~3 weeks in this single recirculating tank, then they moved to their separate spawning buckets on May 2nd. Broodstock were bleach-treated to kill worm hitchhikers, this after finding a plethora of arthropod worms pop out of the gigas that co-habitated with my Olys during the pH treatment.

Let’s review the treatment groups:

  • 4 “populations”:
    • 3 distinct F1 populations: Oyster Bay (SS), Dabob Bay (HC), Fidalgo Bay (NF)
    • 1 distinct F2 population: progeny from Oyster Bay F1 (K)
  • 2 temperature treatments & 2 pH treatments per population = 16 broodstock treatment groups
    • SS: 6C+7.2pH, 6C+7.8pH, 10C+7.2pH, 10C+7.8pH x 2 for spawning buckets
    • HC: 6C+7.2pH, 6C+7.8pH, 10C+7.2pH, 10C+7.8pH
    • NF: 6C+7.2pH, 6C+7.8pH, 10C+7.2pH, 10C+7.8pH x 2 for spawning buckets
    • K: 6C+7.2pH, 6C+7.8pH, 10C+7.2pH, 10C+7.8pH
  • Space was limited. I split each SS and NF broodstock treatment group into 2 buckets for spawning but did not split the HC or K groups.

Larval Collection

Eureka! I began observing larvae on May 11th from both K-10deg groups. Because it can take up to 12 days from fertilized eggs to free-swimming larvae, and because all Olys were housed in the same tank until May 2nd, I waited until May 18th to begin rearing any larvae. From May 11-May 18 I collected, counted, and sampled, but did not grow larave (except for system-testing my buckets). Larvae were also collected, counted, and sampled daily (mostly) through July 10th, providing 2 months spawning data. I have ~80 vials of larvae frozen in Rick’s -80 from the following groups:

Treatment Grp # Samples in -80 Total # larvae in samples Ave # larvae in samples
NF-10 Ambient 5 550,120 110,024
NF-10 Low 2 157,117 78,558
NF-6 Ambient 2 146,033 73,017
NF-6 Low 7 752,835 107,548
SN-10 Ambient 13 1,332,420 102,494
SN-10 Low 8 725,307 90,663
SN-6 Ambient 7 947,383 135,340
SN-6 Low 10 1,474,860 147,486
HL-10 Ambient 4 306,610 76,653
HL-10 Low 5 394,313 78,863
HL-6 Ambient 1 39,400 39,400
HL-6 Low 3 553,900 184,633
K-10 Ambient 3 27,843 9,281
K-10 Low 3 338,017 112,672
K-6 Ambient 4 163,777 40,944
K-6 Low 5 190,357 38,071

Larval Stocking

May 21st was the first official larval stocking date. Larvae were grown in 5-gallon buckets with 18degC flow-through seawater filtered to 5um, and dosed with live algae cocktail via peristaltic pump to achieve ~100,000 cells/mL. Each bucket was equipped with an air stone, inflowing water at 8L/hr, and a 100um banjo made from ~6” PVC ring. FSW was heated to ~18degC in a large header tank and distributed to all buckets. New larvae were added to their respective treatment groups’ larval rearing bucket. Buckets were stocked to a maximum density of ~200,000 larvae as per the following calculations (informed by the PSRF & the FAO manual):

  • Rule: 1 larva/mL for 1 tank turnover/day
  • Larvae stocking equation: (24 hrs / (Tank Vol (L) / (flow rate)))1 larvae/mLTank Vol (L) * 1000mL/L = # larvae/mL to stock in bucket
  • Calculation: (24hrs/(15L/8 L/hr))*1 larva/mL * 15 L * 1000 mL/L = 192,000 larvae. I’m going to round up to 200,000 larvae

Check out this video of me collecting newly spawned Oly larvae.


Larval Screening

Twice weekly larval buckets were screened into three size classes: >224um, >180um, >100um. Each size class was subsampled for counts, then the 100um and 180um groups returned to their buckets, and the 224um graduated up to the setting tank. A couple weeks into the larval rearing phase of the experiment, I began noticing high mortality. To cull the dead larvae I instituted a 2-bucket flow-through system, where live larvae were stocked in one tank where the water also entered (the “back” tank), then the live, swimming larvae would get pushed into the “front” bucket and contained via a 100um banjo. This resulted in only dead (mostly) larvae in the back bucket, which was thus defined as the “morts” bucket. During the twice weekly screening day I screened the morts bucket onto 100um only, and counted for #live & #dead.

Larvae Care during metamorphosis

During the twice weekly screening days larvae that held on 224um screen were moved to setting tanks. Setting tanks were inside the hatchery and constituted 180um silos with 18degC FSW dosed with live algae flowing into each silo from 8L/hr drippers. Air stones were positioned in the middle of the setting bucket (not in each silo). Microculch (224um) was sprinkled into each silo, enough to cover the surface area of the silo screen. Three times throughout the experiment contents from the inside setting tanks were moved into setting tanks outside (similar setup, larger tank). Silos were rinsed and soaked in fresh water at 18degC for ~1 minute several times per week.

Raw survival data shows # larvae stocked in each group and % larvae survived to juvenile stage, which were counted by hand/via scope.

Juvenile Care

Juvniles from each group were housed in 450um silos with ~17degC upwelling FSW dosed with live algae concentration via a gravity algae header. Seed was rinsed 1-2 times per week with fresh water. On October 4 seed were moved to screen “packets”, either 450um or 1600um ( epending on whether they could hold on 1600), and affixed to the inside of shellfish cages, then hung off the NOAA dock in Clam Bay. Oysters were rinsed every 7-10 days with fresh water, and inspected for holes in screen.


Side Experiment: South Sound static culture

One day, June 15th, all four treatment groups from the SS (Oyster Bay) F1 population spawned a considerable number of larvae, >75,000. I started a small, separate experiment in static culture, using 1L tri-pours filled to 800mL and stocking 800 larvae/tripour (achieving 1 larvae/mL) in small 100um silos that fit into the tripours. When I initially set the experiment up I only set up 3 replicates per treatment. After conferring with Katherine she suggested increasing the replicates to 5. I did so by evenly distributing all the larvae into 5 tripours/silos. I made water changes daily with fresh FSW pre-mixed with live algae cocktail at 100,000 cells/mL Algae diet was a 1/2 & 1/2 mix of a diatom and flagellate, and concentration was determined via hemocytometer prior to mixing with FSW. Twenty to fifty larvae were sampled from each replicate for imaging to determine growth at days 0, 7, and 14. The remaining larvae were set in their mini-silos via microculch (224um). After day 14 water changes occurred every other day until they were large enough to move into 450um upwelling silos outside. At week 7 I counted the number of juvenline oysters in each silo to determine survival rate.

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Oly 2017 Exp Summary
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