Monday 8/24/15 – Thursday 8/27/15 (Last week of fieldwork!)

Monday 8/24/15

Had a long chat with my advisor, Cathy, about the status of my fieldwork- what data I had collected, what I was leaving behind, plans for a stressor experiment- as well as plans for funding next summer. Puget Sound Restoration Fund has approved me to conduct a common garden experiment next summer with olys from California, Puget Sound, and British Columbia. My hope is to use this rangewide common garden to contextualize the regional-scale one I did this summer.

Tile culling

  • SSA_7: 9
  • SSA_8: 83
  • HCB_1: 160
  • HCB_2: 310
  • HCB_3: 384
  • Took pictures of tiles that were culled last Thursday but didn’t have pictures for.

Growth Rate Experiment

Started taking pictures of larvae from the growth rate experiments conducted earlier in the summer. This was done using an adaptor to attach a phone to the eyepiece of a microscope. Labelling scheme (until I think of a better one) is Population+Replicate_Growth rate experiment (1 or 2)_date sample was taken. Also did live/dead counts of the entire sample.

  • SS3_G1_7/12: 25L/15D
  • SS1_G1_7/12: 57L/8D
  • SS3_G1_7/15: 38L/14D
  • SS2_G1_7/12: 64 L/9D

Tile Set B

  • Cleaned out the tanks from tile set B. Cut off the tiles and randomized them among the wire “cages” in the large tank with the other tiles from Set_A.

Tuesday 8/25/15

Cultch Set

  • SS>450A + SS>450B -> SS> 450B; SS>1000
  • SS_new -> SS>450B; dump
  • HC > 450 ->  HC > 1000
  • HC_new -> HC > 450; dump

Tile culling

  • HC_B_1: 42
  • HC_B_2: 103 from front, 164 from back
  • HC_B_3: 67 front, 42 back
  • HC_B_4: 4
  • HC_B_7: 4 front, 13 back
  • HC_B_8: 12 front, 3 back
  • HC_B_10: 9 back
  • HC_B_11: 1 back
  • HC_B_12: 2 back
  • HC_B_14: 86 front, 5 back
  • SS_B_1: 7 back (none on front)
  • SS_B_2: 4 back (none on front)
  • SS_B_3: 0/3
  • SS_B_7: 0/10
  • SS_B_8: 0/2
  • SS_B_9: 0/5
  • SS_B_10: 0/7
  • SS_B_11: 0/6
  • SS_B_13: 0/1
  • NF_B_1: 0/27
  • NF_B_2: 95/120
  • NF_B_3: 0/13
  • NF_B_4: 77/270
  • NF_B_5: 8/12
  • NF_B_6: 208/92
  • NF_B_7: 0/2
  • NF_B_8: 18/10
  • NF_B_9: 8/19
  • NF_B_10: 70/48
  • NF_B_11: 11/17
  • NF_B_12: 10/27
  • NF_B_13: 1/12
  • NF_B_14: 0/2

Wednesday 8/26/15

  • Dropped off samples and borrowed materials at the Roberts lab.

Thursday 8/27/15

The little oyster babies are leaving the nest for the big open ocean! I started out the day finishing up any culling that was needed. I also looked over previously culled tiles to make sure that at least 1 cm of space was around each oyster.

Culling

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Steven Roberts came to help with the deployment. While I finished up culling, he made labels for the trays but cutting small PVC pipe into 1 inch pieces and etching numbers onto them. We’re also putting waterproof paper in a tube with the tray number.

12 tiles (4 per population) were attached to each tray with zipties. The populations were ordered in the same way for each section of each tray (NF, HC, SS).

Tray with tiles attached

Tray with tiles attached

Pictures were taken of each tile next to a ruler to measure size at deployment of the oysters.

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7 trays were filled up with with tiles. To minimize the effects of location within a stack of trays, we put 4 trays in a stack with a 2′ spacer between the top 2 and bottom 2 trays. An additional tray was used as a cover to keep out predators. We made 2 of these stacks, with an empty tray in Stack 2.

Stack of trays with oyster tiles ready for deployment

Stack of trays with oyster tiles ready for deployment

Order of tiles and trays

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The trays were hung of the dock at Manchester by ~20 foot rope. One of the stacks seemed to float (which was odd), so we tied on clam bags with rocks to both stacks.

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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

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 8/6/15 and Friday 8/7/15

Thursday 8/6/15

Finally had time to collect the new larvae that I’ve been putting back in the buckets. Most were dead, but can still be used to inform how many have been produced over the past few days. Dumped out all of them. Otherwise caught up on counting from Wednesday and cleaning the tanks that were emptied out.

Friday 8/7/15

Started actively randomizing the order of the tanks on the line. While they were always mixed up when cleaning, now I made sure no tank was in the same place 2 cleanings in a row.

Larval tanks

  • NF_Tank2_160 (224) -> 13,300 total: 500 for DNA, rest to NF_SetB
  • NF_Tank2_160 (100) -> spilled ~400 mL on table. Rinsed what larvae I could off of table onto screen. Swimmers added back.
  • HC_Tank2_160 (224) -> 9,000 total: 500 for DNA, rest added to HC_SetB
  • HC_Tank2_160 (100) -> swimmers only
  • SS_Tank2_160 (224) -> 26,425 total, 425 to DNA, 13,000 to SS_SetB, 13,000 to new silo with cultch
  • SS_Tank2_160 (100) -> swimmers only

Tile Set

Total added

  • SS_SetB: 60,500
  • HC_SetB: 54,925
  • NF_SetB; 34,637

Did counts for NF_SetA, HC_SetA, and SS_SetA.

Wednesday 8/5/15

Had meetings today with Brent, Steven, and Ryan to game plan for the rest of my field season (only 23 more days!). Highlights:

  • Wind down animal husbandry
    • Clean out the “New” tanks
    • Stop collecting new larvae
    • Clean out “160” tanks ASAP
    • Transition cultch set care to hatchery staff
  • Take tissue samples from all broodstock for DNA extractions
    • Store half of samples
    • Try to do as many extractions as possible
  • Make a test 2b-RAD library to run on a MiSeq
    • Order reagents for 2b-RAD libraries, decided on 1/4 reduction
  • Prepare tiles to be placed out in Manchester
    • Once they are all set, randomize among tanks
    • Cull to 20-30 per tile to avoid density impacts on growth
    • Zip-tie tiles to trays with cover
  • Data collection from salinity experiment
    • Talk to Ryann about going through salinity samples for a 2nd live/dead count and to take pictures for size measurement

Very productive chat, but made the rest of the day kind of rushed. Did not get around to cleaning the broodstock or collecting new larvae (although I got rid of the “New” tanks so had nowhere to put them). Decided to max out the SetBs after reaching 60,000 or after 1 week.

Larval Tanks

  • NF_Tank2_160 (224) -> 7,931 total: 500 for DNA, rest to NF_SetB
  • NF_Tank2_160 (100) -> swimmers only added back
  • NF_Tank1_new (100) -> DNA taken, rest dumped out
  • NF_Tank1_new (160) -> NF_Tank2_160
  • HC_Tank2_160 (224) -> 14,525 total: 500 for DNA, rest added to HC_SetB
  • HC_Tank2_160 (100) -> swimmers only
  • HC_Tank1_new (100) -> dumped
  • HC_Tank1_new (160) -> HC_Tank2_160
  • SS_Tank2_160 (224) -> 27,300 total, 500 to DNA, rest added to SS_SetB
  • SS_Tank2_160 (100) -> swimmers only
  • SS_Tank1_new (100) -> dumped
  • SS_Tank1_new (160) -> SS_Tank2_160

Tile Sets

  • SS: 47,500
  • HC: 45,925
  • NF: 21,337

Cultch Set

  • Alice showed me how to screen out larger oysters growing on cultch. Very similiar to screening out larvae tanks, except instead of emptying a tank over a screen you dump the silo out over a screen into a filled sink. The oysters that hold on the screen go into a new silo while those that go through are rinsed onto a smaller screen and then put back in the original silo. Sizes go from 450, 1000, 1600…

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