Shelly’s Notebook: Wed. Jul. 24, 2019 Pt. Whitney Juv. Geoduck resistance to stress plans

experimental plans

uc?export=view&id=1Bt66XE4MpND6zPW8NF8JkxBy3fPSF5ac

biological response measurement ideas

options for low pH treatment:

  1. piggy-back on Sam’s treatment: constant low pH 7.0 total scale
  2. variable diel low pH to simulate lagoon conditions

options for high temperature treatment:

  1. constant high temperature of 20C
  2. constant high temperature of 29C (this will likely kill them before 1 month)
  3. variable diel high temperature of 29C to simulate lagoon conditions

Space:

  • There is space avaiable across from our current heath stacks where the totes to put another heath stack in.
  • Matt should have space for 4 trays in some other heath stacks to house any left over animals if needed

NEXT STEPS:

  • begin heath stack set up directly across from heath stacks
    • test out heat control, flow, etc.

Equipment update

  • Thermometer for discrete temp measurements errored out. See slack post. It may just be the probe, but need to test this.
  • Apex status:
    • Controller not lighting up at all when plugged into everything. Need to call support and get help troubleshooting this
    • Power strip lights up fine
    • Aqua buses:
      • how many can be linked to 1 Apex controller?
    • Probes:
      • ideally need 12 pH + temp sets
    • brought Controller back to UW to troubleshoot
  • Have 20 total heath trays plumbed for downwelling
  • Can use 1 micron screen mesh tray inserts with large grain sand (Matt has extra).
    • NEED to check:
      • # of good quality inserts we have (ideally 10)
      • # of inserts that need dividers added
        • can cut dividers from black plexiglass upstairs in the hatchery
  • Controlled heating source:
    • NEED 2 heat rods that can be plugged into the apex
      • have these at UW, need to test
  • Flow:
    • if doing constant pH, NEED 4 pumps with adjustable flow if using same header conicals as Sam
    • if doing diel variable pH, use different conical and don’t need adjustable flow
      • extra pumps at hatchery (imagitarium secondary pumps used for circulation in broodstock experiment)
      • would need additional probe set
  • More equipment will free up when Sam is done by the end of August

Checked on animals

uc?export=view&id=1Pe-WvIdQ4T7YklqcTAnnKUcnp6suxDSZ

Size difference between H2T3 animals and H2T1 animals from cascade feeding uc?export=view&id=1D_90h2WL1ItCFDRABYHSHBRVeJdi6lrL

In-flow hose was not going directly into the heath tray insert in H2T1: uc?export=view&id=1igdszlxJwQZ6NFHv9yTogQA8WAY3lwNt

Tried to break up algae and clear mesh to improve flow of food and shuffled trays around to new order:

  1. H2T1
  2. H2T5
  3. H2T7
  4. H2T3
  5. H2T6 (extras)
  6. H2T2 (low density)

*only swapped H2T3 with H2T1 because these trays showed the biggest size differences.

*Left H2T6 and H2T2 in same positions because they had a lot of die off and aren’t going to be part of the experiment.

Size data (photos) for all animals are here. Size ranges for all are about 2-6mm. I will do individual measurements with image J

Survival and size data for larval rearing through now I think is too convoluted to draw conclusions from parental exposure.

Prior to pH x temp experiment, I can size select to start with animals of similar sizes across the board.

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Laura’s Notebook: Oly RNA isolation – larvae

July 23, 2019

Homogenized and frozen larvae to prep for RNA isolation, and aliquoted larve in ethanol for imaging/measuring at a later date. These larvae were collected during spring 2017, captured on screens upon release from mother, concentrated into microcentrifuge tubes, and placed immediately into a -80 freezer to preserve. Larvae in the same tube are likely to be full siblings or half siblings; the number of larvae collected on that day / from that collection bucket is a good indication of whether more than one female released brood that day (we expect ~200k larvae/female).

First step is homogenization, which is particularly necessary due to larvae having shells that need to be broken up. As with the ctenidia samples, I used mortar+pestle and liquid nitrogen. I pre-portioned 1 mL of RNAzol into microcentrifuge tubes, and transferred up to 100 mg homogenized larvae into chilled RNAzol. NOTE: I tried to get as much tissue as possible up to 100 mg, since the larval shells likely comprise a significant percentage of the mass.

In total I have 83 larval samples. I isolated RNA from 14 of these samples in spring 2018 for the test QuantSeq round. A few resulted in too little RNA, 2 of which had sufficient larvae remaining for another attempt to isolate RNA. So, I homogenized a total of 70 samples into RNAzol in 10 batches (~4 days of work), with an additional 2 controls (just liquid nitrogen ground into mortar+pestle).

July 24, 2019

Did a first round of RNA isolation (n=12, batch #1) to test protocol. Followed RNAzol protocol like with the ctenidia samples, with two exceptions: 1) after addition of DEPC-treatetd water (0.2 mL), centrifuged at 16,000 rcf (not 12,000); 2) added 100 uL DEPC-treated water in last step to dissolve RNA. Samples in this 1st batch: ABCDEFG

Also of note: after precipitating the DNA/proteins, the supernatant retained a black/gray color. Then, after adding isopropanol, the RNA pellet was predominantly black in 10 of the 12 samples (the 2 samples with small, white pellets were X and Y). Then, upon dissolving RNA in the DEPC-treated water and vortexing for 5 minutes, there was a black substance that settled into the bottom of the tubes. A black substance occurred before, in 2018 when I did a preliminary round of RNA isolation from the same study’s Oly larvae (see notebook post); this didn’t seem to interfere with the successful QuantSeq run.

I quantified RNA concentration in this first batch, using 1 uL of the RNA solution, pulled from the surface of the solution to avoid the black substance. Measured RNA in all samples, at a concentration of between 30-90 ng/uL.

Image of a few tubes showing that RNA pellete included black substance: IMG_8758

Image of RNA dissolved in water, showing a black substance settled at the bottom of the tubes. IMG_8757

July 26, 2019

Big day of RNA isolation – did 4 batches of 12, but processed 2 batches simultaneously, offset by about 10 minutes. This was feasible because there is down time during reactions/centrifuging.

Batches #2 and #3 (run simultaneously offset), followed the same protocol as with batch #1 (above), with one exception: added 75 uL of DEPC-treated water in final step to dissolve RNA.

Batch #2 samples: 402, 412, 421, 431, 432, 442, 452, 461, 462, 482, 532, 542

Batch #3 samples: 403, 404, 472, 473, 483, 484, 491, 522, 533, 552, 562, 571

Batches #4 and #5 (run simultaneously, offset), followed same protocol as batches 2 & 3, with one exception: did not vortex homogenate prior to transferring 500 uL to new tubes with the 200 uL DEPC-treated water. The resulting RNA dissolved in water had significantly less black substance, likely due to this adjustment.

I quantified RNA isolated from all samples, shown in the below table, and the spreadsheet is saved in the repo. A handful of samples resulted in relatively poor yield (452, 461, 462, 472, 533, 552), and several samples had a substantial amount of black substance mixed into the RNA solution (404, 431, 432, 442, 443(ish), 461, 462, 472, 486(ish), 521(ish), 522(ish), 552). Some samples in these these two categories overlapped – 461, 462, 472, 552. I will process some of the remaining homogenate (250 uL) to try to get a cleaner batch of RNA.

Date larvae collected Cohort Treatment TISSUE SAMPLE # HOMOG. TUBE # VOL RNAzol (mL) MASS TISSUE (mg) DATE HOMOG. HOMOG. BATCH RNA ISOLATION DATE RNA ISOLATION BATCH Total RNA volume remaining, uL (RNA + H2O) [RNA] ng/uL Amount of RNA (ng) Volume needed for 500 ng RNA Notes
5/24/17 Dabob Bay 10 Ambient 14-A 401 1 100 19-Jul 1 24-Jul 1 99 52.0 5,148 9.62
5/31/17 Dabob Bay 10 Ambient 31-A 402 1 10 20-Jul 3 26-Jul 2 74 140.0 10,360 3.57
6/19/17 Dabob Bay 10 Ambient 75-A 403 1 40 22-Jul 5 26-Jul 3 74 148.0 10,952 3.38
6/29/17 Dabob Bay 10 Ambient 80-A 404 1 110 22-Jul 6 26-Jul 3 74 95.2 7,045 5.25
5/26/17 Dabob Bay 10 Low 23-A 411 1 10 19-Jul 1 24-Jul 1 99 57.2 5,663 8.74
5/27/17 Dabob Bay 10 Low 27-A 412 1 10 20-Jul 3 26-Jul 2 74 60.8 4,499 8.22
6/10/17 Dabob Bay 10 Low 58-A 413 1 80 22-Jul 7 26-Jul 4 74 91.4 6,764 5.47
6/12/17 Dabob Bay 10 Low 60-A 414 1 20 23-Jul 8 26-Jul 4 74 136.0 10,064 3.68
6/12/17 Dabob Bay 6 Ambient 59-A 421 1 10 20-Jul 2 26-Jul 2 74 43.0 3,182 11.63
6/7/17 Dabob Bay 6 Low 51-A 431 1 20 20-Jul 2 26-Jul 2 74 43.4 3,212 11.52
6/17/17 Dabob Bay 6 Low 72-A 432 1 50 22-Jul 4 26-Jul 2 74 47.6 3,522 10.50
6/17/17 Dabob Bay 6 Low 73-A 433 na na na na na na na na na na
6/19/17 Dabob Bay 6 Low 74-A 434 1 60 23-Jul 9 5 74 67.8 5,017 7.37
5/25/17 Fidalgo Bay 10 Ambient 20-A 441 1 70 19-Jul 1 24-Jul 1 99 46.0 4,554 10.87
6/3/17 Fidalgo Bay 10 Ambient 38-A 442 1 80 20-Jul 3 26-Jul 2 74 LOW
6/7/17 Fidalgo Bay 10 Ambient 53-A 443 1 60 22-Jul 6 26-Jul 4 74 58.4 4,322 8.56
6/14/17 Fidalgo Bay 10 Ambient 63-A 444 1 80 22-Jul 7 26-Jul 4 74 91.2 6,749 5.48
6/15/17 Fidalgo Bay 10 Ambient 65-A 445 1 40 23-Jul 8 26-Jul 5 74 132.0 9,768 3.79
5/24/17 Fidalgo Bay 10 Low 16-A 451 1 70 19-Jul 1 24-Jul 1 99 68.4 6,772 7.31
5/24/17 Fidalgo Bay 10 Low 18-A 452 1 80 20-Jul 3 26-Jul 2 74 37.8 2,797 13.23 poor yield b/c black substance w/ RNA?
6/3/17 Fidalgo Bay 10 Low 36-A 453 1 80 23-Jul 9 27-Jul 6 74 85.2 6,305 5.87
5/26/17 Fidalgo Bay 6 Ambient 22-A 461 1 100 20-Jul 2 26-Jul 2 74 31.0 2,294 16.13 poor yield b/c black substance w/ RNA?
5/29/17 Fidalgo Bay 6 Ambient 29-A 462 1 60 22-Jul 4 26-Jul 2 74 29.8 2,205 16.78 poor yield b/c black substance w/ RNA?
5/25/17 Fidalgo Bay 6 Low 19-A 471 1 100 20-Jul 2 24-Jul 1 99 33.8 3,346 14.79
5/26/17 Fidalgo Bay 6 Low 21-A 472 1 70 22-Jul 4 26-Jul 3 74 8.3 613 60.39 poor yield b/c black substance w/ RNA?
6/5/17 Fidalgo Bay 6 Low 46-A 473 1 50 22-Jul 5 26-Jul 3 74 120.0 8,880 4.17
6/5/17 Fidalgo Bay 6 Low 47-A 474 1 50 22-Jul 6 26-Jul 4 74 81.0 5,994 6.17
6/6/17 Fidalgo Bay 6 Low 50-A 475 1 80 22-Jul 7 26-Jul 4 74 110.0 8,140 4.55
6/10/17 Fidalgo Bay 6 Low 54-A 476 1 80 23-Jul 8 26-Jul 5 74 43.4 3,212 11.52
6/19/17 Fidalgo Bay 6 Low 76-A 477 1 40 23-Jul 9 27-Jul 6 74 HIGH
5/20/17 Oyster Bay C1 10 Ambient 02-A, 02-B 481 1 40 19-Jul 1 24-Jul 1 99 64.4 6,376 7.76
5/20/17 Oyster Bay C1 10 Ambient 04-A, 04-B 482 1 60 20-Jul 3 26-Jul 2 74 67.2 4,973 7.44
5/21/17 Oyster Bay C1 10 Ambient 03-A, 03-B 483 1 110 22-Jul 4 26-Jul 3 74 95.8 7,089 5.22
5/23/17 Oyster Bay C1 10 Ambient 09-A 484 1 40 22-Jul 5 26-Jul 3 74 66.2 4,899 7.55
6/1/17 Oyster Bay C1 10 Ambient 34-A 485 1 20 22-Jul 6 26-Jul 4 74 156.0 11,544 3.21
6/3/17 Oyster Bay C1 10 Ambient 39-A 486 1 90 22-Jul 7 26-Jul 4 74 41.0 3,034 12.20
6/3/17 Oyster Bay C1 10 Ambient 40-A 487 1 30 23-Jul 8 26-Jul 5 74 112.0 8,288 4.46
6/4/17 Oyster Bay C1 10 Ambient 44-A 488 1 70 23-Jul 9 26-Jul 5 74 57.2 4,233 8.74
6/6/17 Oyster Bay C1 10 Ambient 49-A 489 1 10 23-Jul 9 27-Jul 6 74 57.2 4,233 8.74
6/14/17 Oyster Bay C1 10 Ambient 64-A 490 1 70 22-Jul 7 27-Jul 6 74 58.6 4,336 8.53
6/15/17 Oyster Bay C1 10 Ambient 66-A 491 1 20 22-Jul 5 26-Jul 3 74 126.0 9,324 3.97
7/6/17 Oyster Bay C1 10 Ambient 81-A 492 1 70 22-Jul 6 26-Jul 5 74 122.0 9,028 4.10
5/21/17 Oyster Bay C1 10 Low 06-A, 06-B 501 1 <10 19-Jul 1 na na na na na na
5/23/17 Oyster Bay C1 10 Low 08-A 502 na na na na na na na na na na
5/26/17 Oyster Bay C1 10 Low 24-A 503 na na na na na na na na na na
5/27/17 Oyster Bay C1 10 Low 26-A 504 na na na na na na na na na na
5/31/17 Oyster Bay C1 10 Low 32-A 505 na na na na na na na na na na
6/14/17 Oyster Bay C1 10 Low 62-A 506 1 80 20-Jul 2 24-Jul 1 99 63.8 6,316 7.84
6/15/17 Oyster Bay C1 10 Low 67-A 507 na na na na na na na na na na
6/24/17 Oyster Bay C1 10 Low 79-A 508 na na na na na na na na na na
5/23/17 Oyster Bay C1 6 Ambient 10-A 511 na na na na na na na na na na
6/3/17 Oyster Bay C1 6 Ambient 37-A 512 na na na na na na na na na na
6/5/17 Oyster Bay C1 6 Ambient 45-A 513 30 23-Jul 10 26-Jul 5 74 156.0 11,544 3.21
6/6/17 Oyster Bay C1 6 Ambient 48-A 514 na na na na na na na na na na
6/15/17 Oyster Bay C1 6 Ambient 69-A 515 na na na na na na na na na na
6/17/17 Oyster Bay C1 6 Ambient 71-A 516 na na na na na na na na na na
6/19/17 Oyster Bay C1 6 Ambient 77-A 517 na na na na na na na na na na
5/21/17 Oyster Bay C1 6 Low 01-A, 01-B- 01-C 521 1 70 20-Jul 2 24-Jul 1 99 54.4 5,386 9.19
5/22/17 Oyster Bay C1 6 Low 07-A 522 1 20 22-Jul 5 26-Jul 3 74 60.8 4,499 8.22
5/27/17 Oyster Bay C1 6 Low 25-A 523 1 30 22-Jul 6 26-Jul 4 74 60.6 4,484 8.25
5/27/17 Oyster Bay C1 6 Low 28-A 524 1 80 22-Jul 7 26-Jul 4 74 80.8 5,979 6.19
5/29/17 Oyster Bay C1 6 Low 30-A 525 1 30 23-Jul 8 26-Jul 5 74 128.0 9,472 3.91
5/31/17 Oyster Bay C1 6 Low 33-A 526 1 30 23-Jul 9 27-Jul 6 74 65.2 4,825 7.67
6/14/17 Oyster Bay C1 6 Low 61-A 527 1 90 22-Jul 6 27-Jul 6 74 81.4 6,024 6.14
6/15/17 Oyster Bay C1 6 Low 68-A 528 1 30 23-Jul 8 26-Jul 5 74 162.0 11,988 3.09
6/17/17 Oyster Bay C1 6 Low 70-A 529 1 70 23-Jul 9 26-Jul 5 74 73.4 5,432 6.81
5/24/17 Oyster Bay C2 10 Ambient 17-A 531 1 60 19-Jul 1 24-Jul 1 99 88.2 8,732 5.67
6/3/17 Oyster Bay C2 10 Ambient 42-A 532 1 40 20-Jul 3 26-Jul 2 74 158.0 11,692 3.16
6/10/17 Oyster Bay C2 10 Ambient 56-A 533 1 <10 22-Jul 4 26-Jul 3 74 7.4 548 67.57 poor yield b/c very little tissue (likely)
5/23/17 Oyster Bay C2 10 Low 12-A 541 1 40 10-Jul 1 24-Jul 1 99 45.6 4,514 10.96
5/24/17 Oyster Bay C2 10 Low 13-A 542 1 30 20-Jul 3 26-Jul 2 74 82.0 6,068 6.10
6/4/17 Oyster Bay C2 10 Low 43-A 543 1 80 22-Jul 7 27-Jul 6 74 61.4 4,544 8.14
6/1/17 Oyster Bay C2 6 Ambient 35-A 551 1 30 20-Jul 2 24-Jul 1 99 86.0 8,514 5.81
6/3/17 Oyster Bay C2 6 Ambient 41-A 552 1 80 22-Jul 5 26-Jul 3 74 17.5 1,295 28.57 poor yield b/c black substance w/ RNA?
6/10/17 Oyster Bay C2 6 Ambient 55-A 553 1 30 23-Jul 8 26-Jul 4 74 200.0 14,800 2.50
6/20/17 Oyster Bay C2 6 Ambient 78-A 554 1 30 23-Jul 9 26-Jul 5 74 156.0 11,544 3.21
5/21/17 Oyster Bay C2 6 Low 05-A 561 1 40 20-Jul 2 24-Jul 1 99 43.4 4,297 11.52
5/23/17 Oyster Bay C2 6 Low 11-A 562 1 90 22-Jul 5 26-Jul 3 74 106.0 7,844 4.72
5/24/17 Oyster Bay C2 6 Low 15-A 563 1 50 22-Jul 6 26-Jul 4 74 84.6 6,260 5.91
6/7/17 Oyster Bay C2 6 Low 52-A 564 1 not recorded 22-Jul 7 27-Jul 6 74 HIGH
6/10/17 Oyster Bay C2 6 Low 57-A 565 1 10 23-Jul 8 27-Jul 6 74 31.8 2,353 15.72
NA RNA Control RNA Control 571 1 10 22-Jul 4 26-Jul 3 74 LOW NA NA
NA RNA Control RNA Control 572 1 10 23-Jul 10 26-Jul 5 74 LOW NA NA
NA RNA Control RNA Control 574 NA NA NA NA 27-Jul 6 74 LOW NA NA

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