Friday 11/6/15

At 9:00am I turned off the chiller and took a sample of oysters from the overnight treatment. It appeared that many of the oysters from the overnight treatment were gaping slightly, but they still offered resistance during dissection. I dissected 6 cultch set oysters per population and 4 tile set oysters and put the whole body tissue in RNALater. I also dissected 10 oysters per population from the control treatment. Instead of moving the overnight oysters over to the control tank, I let the temperature gradually increase by draining some of the tank and adding ambient water. We switched the control tank over to a flow-through system with live algae dripping in. At 2:00pm I moved the overnight chilled oysters to the control tank. The hatchery folks are going to keep an eye on the oysters for 2 weeks, after which they’ll sample all of the survivors.

Time
9:00am
-control tank: 9degC
-chilled tank: 0degC; turned off chiller
10:30am
– chilled tank: 3degC
12:00pm
-chilled tank: 5degC
1:00pm
-chilled tank: 7degC
-control tank: 10degC; switched to flowthrough system
2:00pm
-chilled tank: 9degC; added overnight oysters to control tank
-control tank: 11degC

I took apart the tile trays and drained out the tank they had been in. Some tiles that had only a few oysters on them were not used in the temperature experiment. I dissected these and put the whole body in RNALater, figuring they could be used for genotyping or looking at epigenetic in the F2 generation. Some of them I took a picture for size data and some of them I measured with a ruler.

Samples from tiles:

  1. SSB10
  2. NFA7_1
  3. NFA7_2
  4. SSB11
  5. NFA13R: 1.3cm x 1.4cm
  6. HCA10_1: 1.3 x 1.2
  7. HCA10_2: .9 x .7
  8. HCA15 (top)
  9. HCA6 (top)
  10. NFB12 (middle)
  11. NFB13R (2nd from top)
  12. NFB7 (left)
  13. HCA8
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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)

A taste of data analysis

Playing around with some data on protoshell length taken by Ryann (PSRF intern) from the salinity experiment.

In R:

 > plot(Length~Population, data = sal30df)
sal30_length

Boxplot of protoshell length in 7 day old Olympia oyster larvae from 3 populations in Puget Sound. n = 20 per population

Did an anova:

> aov.sal30 <- aov(Length~Population, data=sal30df)
> summary(aov.sal30)

Df Sum Sq Mean Sq F value Pr(>F)
Population 2 9281 4641 14.81 6.61e-06 ***
Residuals 57 17861 313

Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Indicates that the means of protoshell length are significantly different (yay!).

> pop <- sal30$Population
> len <- sal30$Length
> ptest <- pairwise.t.test(len, pop, p.adjust = "bonferroni")

Pairwise comparisons using t tests with pooled SD

data: len and pop

Fidalgo Bay Hood Canal
Hood Canal 0.0913             –
Oyster Bay 0.0069          3.9e-06

P value adjustment method: bonferroni

Very significant p-value between Hood Canal and Oyster Bay.

Wednesday 9/23/15

  • Did more extractions of broodstock samples. I think once this test library is done the broodstock libraries should be 1st priority, with samples from the stressor experiment 2nd and other larvae samples 3rd.
    • Used the EZNA kit with a 4 hour digestion time. All the tissue for HC1_16 was used up.
  1. HC1_11
  2. HC1_12
  3. HC1_13
  4. HC1_14
  5. HC1_15
  6. HC1_16
  7. HC1_17
  8. HC1_18
  9. HC1_19
  10. HC2_1
  11. HC2_2
  12. HC2_3
  13. HC2_4
  14. HC2_5
  15. HC2_6
  16. HC2_7
  17. HC2_8
  18. HC2_9
  19. HC2_10
  20. HC2_11
  21. HC2_12
  22. HC2_13
  23. HC2_14
  24. HC2_15
  • Finished the ethanol precipitation started on Tuesday 9/22/15. Left samples in 10 uL of water in 4degC overnight.
  • Qubit of samples for phylogeographic study. Made list of samples for GBS library.
      1. BC1_12: 25.2
      2. BC1_8: 6.44 (8)
      3. BC2_7: 10.6
      4. BC2_13: 11.5
      5. BC2_9: 13 (8)
      6. BC3_13: 108
      7. BC3_12: 15 (8)
      8. BC4_2: 8.84
      9. BC4_17: 11.4 (8)
      10. CA1_18: 24.4
      11. CA1_1: 36
      12. CA1_2: 17.4 (8)
      13. CA2_9: 43.8
      14. CA2_12: 18.8 (5)
      15. CA3_6: 62.3 (5)
      16. CA4_1: 5.1
      17. CA4_7: 19.8 (8)
      18. CA5_10: 48.8 (8)
      19. CA6_8: 5.14
      20. CA6_15: 25.2 (7)
      21. CA7_11: 7.4
      22. CA7_16: 19.1
      23. CA7_15: 14.9 (8)
      24. OR1_ 1: 7.66
      25. OR1_5: 29.2 (7)
      26. OR2_6: 12.9
      27. OR2_12: 15.8
      28. OR2_15: 18.2 (7)
      29. OR3_15: 9.24
      30. OR3_20: 13.7 (7)
      31. WA10_16: 25.4
      32. WA10_13: 9.52 (8)
      33. WA11_10: 18.7
      34. WA11_22: 17.4
      35. WA11_4: 11.7
      36. WA11_17: 4.78 (8)
      37. WA12_15: 24.4
      38. WA12_11: 4.18  (8)
      39. WA13_12: 7.2
      40. WA13_5: 4.4
      41. WA13_15: 45.1 (9)
      42. WA9_2: 11.4 (7)
      43. WA1_16: 62 (8)
      44. Conch_1: 12.4
      45. conch_2: 12.3
      46. conch_4: 9.65
      47. CA5_12: 21.8 (repeat with 2)
      48. CA5_10 repeat within

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

Monday 8/10/15 and Tuesday 8/11/15

Monday 8/10/15

Larvae tanks

  • NF_Tank2_160 (224) -> 10,500 total: 500 for DNA, rest to NF_SetB
  • NF_Tank2_160 (100) -> swimmers only
  • HC_Tank2_160 (224) -> 9,187 total: 500 for DNA, 5,187 added to HC_SetB, rest to cultch set
  • HC_Tank2_160 (100) -> swimmers only
  • SS_Tank2_160 (224) -> 11,900 total, added to SS cultch
  • SS_Tank2_160 (100) -> swimmers only

Tile Set

  • Did live/dead counts for both A and B
  • Rinsed tiles (have been doing this everyday I’m in)
  • New totals
    • NF_SetB: 45,137
    • HC_SetB: 60,000

New larvae

  • no new larvae

Tuesday 8/11/15

Animal husbandry

  • Cleaned broodstock buckets
  • Rinsed cultch and tiles

Lab work

Extracted DNA from 24 larvae samples that would be good candidates for test 2b-RAD libraries. I chose sets of new larvae (“LC” for larvae catch), “160”s, and “224”‘s with 3-5 days in between.

Population Tank Family Size Date Storage Est. # Date extracted
1 Hood Canal LC >100 7/13/2015 75/95 EtOH 8/11/2015
2 Fidalgo Bay NA LC 100 7/13/2015 75% EtOH 8/11/2015
3 South Sound NA LC 100 7/13/2015 RNALater 8/11/2015
4 Hood Canal NA HC2 100 7/17/2015 RNALater 8/11/2015
5 Hood Canal NA LC 100 7/17/2015 RNALater 8/11/2015
6 South Sound NA LC 100 7/17/2015 RNALater 8/11/2015
7 Hood Canal NA LC 100 7/20/2015 RNALater 8/11/2015
8 Fidalgo Bay NA LC 100 7/20/2015 RNALater 8/11/2015
9 South Sound NA LC 100 7/20/2015 RNALater 8/11/2015
10 Hood Canal HC_Tank1_160 NA 160 7/20/2015 RNALater 8/11/2015
11 Fidalgo Bay NF_Tank1_new NA 160 7/20/2015 RNALater 8/11/2015
12 South Sound SS_Tank1_new NA 160 7/15/2015 RNALater 8/11/2015
13 Hood Canal HC_Tank1_new NA 160 7/24/2015 RNALater 8/11/2015
14 Fidalgo Bay NF_Tank1_new NA 160 7/24/2015 RNALater 8/11/2015
15 South Sound SS_Tank1_new NA 160 7/24/2015 RNALater 8/11/2015
16 Hood Canal HC_Tank1_new NA 160 7/27/2015 RNALater 8/11/2015
17 Fidalgo Bay NF_Tank1_new NA 160 7/27/2015 RNALater 8/11/2015
18 South Sound SS_Tank1_new NA 160 7/27/2015 RNALater 8/11/2015
19 Hood Canal HC_Tank2_160 224 8/3/2015 RNALater 8/11/2015
20 Fidalgo Bay NF_Tank2_160 224 8/3/2015 RNALater 8/11/2015
21 South Sound SS_Tank2_160 224 8/3/2015 RNALater 8/11/2015
22 Hood Canal HC_Tank2_160 >224 8/7/2015 RNALater 350 8/11/2015
23 Fidalgo Bay NF_Tank2_160 >224 8/7/2015 RNALater 504 8/11/2015
24 Oyster Bay SS_Tank2_160 >224 8/7/2015 RNALater 641 8/11/2015

I still have to go back over my notes to get estimated number for some of them. Storage was mostly in RNALater. A lot of the samples had some white precipitate on the bottom. If larvae weren’t  in the precipitate I sucked it out before adding the lysis buffer/proteinase K. Also had the same issue previously with larvae being buoyant in the RNALater even after a spindown. Halfway through trying to siphon off as much liquid as I could, I did some research and found that addition of some ice-cold PBS will change the density of the liquid and allow the larvae to settle out. This worked really well and was done for 2,10,13,15,19,20,21,24. It also dissolved most of the white precipitate.

Followed the protocol I listed here, with a 2.5 hour digest. Could not findd the gel rig set-up (found out later it’s in a different building).

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

Friday 7/31/15

Nine larvae/setting tanks to clean! Michael and I had an evening flight out east for his cousin’s wedding, so today was a pretty rushed day trying to get everything done and still make the 4:05p ferry.

Larvae tanks

  • NF_Tank2_160 (224) -> 31,200 total: 500 for DNA, 11,000 to cultch set B, 19,700 dumped out
  • 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) -> 17,281 total: 500 for DNA, 11,000 added to cultch set B, 5,781 added back to HC_Tank2_160
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> swimmers only
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 11,537 total, 537 to DNA, 11,000 to cultch set B
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> swimmers only
  • SS_Tank1_new (160) -> SS_Tank2_160

Cultch Setting System

Set up a 2nd silo replicate for all 3 populations. As SS only had ~11,000 224-sized larvae, I added 11,000 larvae to each of the silos. 5,781 extra HC larvae were added back to HC_Tank2_160. With NF, there were ~9,000 dead 224-sized larvae- most likely due to my adding the the ones I wasn’t using back in to Tank2_160. If a larva that is ready to set can’t find an appropriate surface, it will eventually die. I dumped out the 19,700 larvae that were not used in the cultch set (see previous post for my regret about this).

New larvae

Only saw some larvae in HC and SS. I ran out of time to take samples for counting, so just screened out the larvae, cleaned the broodstock buckets, and dumped the larvae back into the buckets.