Wednesday 8/19/15 and Thursday 8/20/15

Wednesday 8/19/15

There was a silo making party at the hatchery today for the rock scallop project. With little animal husbandry to do, I decided to do a DNA extraction and help out making silos while the tissue was digesting.

Tile Culling

  • HCA_7: 33 removed
  • NFA_1: 123
  • NF_A_2: 134
  • NF_A_3: 102
  • NF_A_4: 32

Cultch Set

NF_A + NF_New -> NF> 450_B; NF_New

DNA Extraction

Took tissue out of RNALater onto sterile weigh boat and cut off ~30 mg of tissue with scalpel (have previously weighed out chunks of adductor muscle tissue to help eyeball this amount). Replaced rest of tissue in RNALater. Wiped scalpel and forceps with 85% ethanol and sterilized over benchtop bunsen burner in between samples. Otherwise, followed EZNA protocol as described in a previous post.

  1. SS2_1
  2. SS2_2
  3. SS2_3
  4. SS2_4
  5. SS2_5
  6. HC1_1
  7. HC1_2
  8. HC1_3
  9. HC1_4
  10. HC1_5
  11. NF1_1
  12. NF1_2

Thursday 8/20/15

Tile Set B

  • bleached line
  • cleaned tanks
  • did counts for HC_SetB and NF_SetB

Tile Set A

Had a discussion with Ryan on the ferry about outplanting my tiles. He recommended transitioning the setters slowly to ambient temperature (they are still on heated at 18degC). My room at the hatchery is not really set up for this, so Alice helped me set up one of the large tanks outside that has a heated and an ambient spigot. I tried to just set the tiles on the large 400um mesh screens, but they did not all fit so I just randomized the tiles among 3 poultry wire setups (the same that were in the setting system tanks). The spigots are at one end of the tank and the outflow is at the other, promoting water flow. Food is splashed in by hand and then added in through a dropper. Some of the outflow is actually pumped back in to the side of the tank with the tiles to reduce algae waste.

Tile culling

  • NFA_5: 29 removed
  • SSA_3: 15
  • SSA_5: 61
  • SSA_2: 10
  • SSA_6: 13

Cultch Set

  • NF >1000 -> NF >1600; NF> 1000
  • NF > 450 ->  NF > 1000; NF > 450
  • HC > 1000 -> HC > 1600; HC > 1000
  • HC> 450  ->  HC > 1000; HC > 450
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Sunday 8/16/15 – Tuesday 8/18/15

Sunday 8/16/15

Came in on a Sunday to knock out some more dissections and transition to a Sunday/Tuesday/Thursday cleaning for this week since I’d be out of town on Friday.

Dissections

  • NF3 and NF5

Husbandry

  • Bleached the line
  • Rinsed tiles and cultch
  • Cleaned all of the A tanks, HC_SetB, and NF_SetB
  • Counts for HC_SetB and NF_SetB

Monday 8/17/15

Cultch Set

  • HC SetA + HC Set New -> HC >450; HC Set New
  • HC > 450 -> HC >1000; HC > 450

Dissections

  • HC1, HC2, HC2, HC3

Husbandry

  • rinsed tiles and cultch
  • cleaned SS_SetB tank

Tile Culling

Started culling for density off of tiles from A sets that were very crowded. Using a clean scalpel, I would remove spat so that only ~30 were left on a side of a tile. I put the spat in a 1.5 mL tube, rinsed with freshwater Millepore water, then stored in RNALater. I kept track of how many were taken from a tile, but in some cases it is an estimate when I was scraping off many that had settled on top of each other. My primary goal was to reduce the time the tile was out of the water.

  • HC_SetA_1: 300 removed
  • HC_SetA_2: 160
  • HC_SetA_3: 17 from front, 8 from back
  • HC_SetA_4: 420 from front

Tuesday 8/18/15

Dissection

  • HC4, HC5 (finished!)

Husbandry

  • Bleached the line
  • Cleaned all tanks
  • Counts for B set

Cultch Set

  • SS_SetA + SS_Set_New -> SS >450; SS_Set_New (on 1000 cultch)

Tile Culling

  • HC_SetA_4: 400 front
  • HC_SetA_5: 150
  • HC_SetA_6: 76

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).

Thursday 7/30/15 (Lab work!)

I got to do some bona fide lab work today, which was a nice change of pace. I’ve been taking samples of larvae for DNA sequencing at various points throughout the experiment:

  • From all newly released larvae (either from each family or combined, depending on how I filtered them out)
  • From larvae in the “New” tanks that reach 160 microns
  • From larvae in the “160” tanks that reach 224 in size
  • Occasionally pooled larvae from a tank

These samples have mostly been stored at -20degC in .5-1 mL of RNALater, but duplicates of many were also stored in ethanol (1st in 75%, then in 95%). Earlier in the summer I wanted to do a test extraction to see if there was a particular storage method that worked best and figure out which extraction kit to use, but then the oysters needed maintenance 6 days a week and all of a sudden in was almost August. With the growth rate experiments and larval production essentially done, I finally had a day to do the test extraction.

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