https://docs.google.com/spreadsheets/d/1mw23331tl9nfIdA8k9EL3kTSPXu0gAqVovCFrMip1Ac/edit?usp=sharing

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.

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…

Friday 6-26-15

Larvae in catch buckets:

• Some: HC3, HC2(maybe), HC4, NF5, HC1, SS5, SS1 (maybe)
• Lots: NF1, NF2 (bottom), SS2, SS4
• Added to NF_Tank1, SS_Tank2, and HC_Tank2
• Combined all the families from a group together again for screening and counting.
• I also screened out the buckets that the broodstock are kept in while I’m cleaning. All of them had a few thousand larvae in them, and these were added to the appropriate tank.

Larval tank counts:

• SS_Tank1: over a 140 screen and a 100 screen. Needed to use 2 beakers for the 140 as there were so many. Most of the larvae that were on the 100 screen were dead, so I did not add these back to the tank as they may make the rest of the larvae sick.
• HC_Tank1: screened them over a 160 micron screen and a 100 micron screen, as this tank has the oldest larvae. I was excited to see that some of them had indeed grown up, as about half of the larvae held on the 160 screen.
• NF_Tank1
• While taking the 1 mL samples from the SS_Tank1 beakers, I noticed the pipet tip did not fill up to the 1 mL mark. This is a PSRF pipet, and an older model. I compared the pipet to another one from the molecular lab I knew to be calibrated, and found that the PSRF pipet consistently gave .8 mL instead of 1 mL. This was the same pipet I used on Thursday and the same one Steven used on Wednesday. I’ll ask him if he noticed the pipet tip looking low, but for now I’ve changed the spreadsheet from those 2 days to reflect a drop size of .8.

Larval count data

Cleaning: had Natalie help clean some of the broodstock buckets.

I did not enter the data from my waterproof notebook into my spreadsheet until Sunday. This is when I noticed I had gained ~5,000 larvae in NF_Tank1. I

Sunday 6-28-15

I came in Sunday planning to shift some of the M-W-F cleaning to Sat/Sun-Tues-Thurs so that I have more time for taking samples and setting up the next stage. Unfortunately, the hot water went down again for a few hours which delayed cleaning. I wasn’t able to filter out the newly spawned larvae, but I did filter out all the 100 L larval tanks and counted them.

Observed larvae:

• Some: NF1, HC3
• Lots: SS1, HC5, SS2, SS3, HC4, SS4

Went to the hatchery in the morning to check for larvae, clean/replace banjo filters, rinse drippers, and replenish algae. As there was a hatchery-wide mortality event after the temperature drop over the weekend, I started 2 new 100 L larval tanks: SS_Tank2 and HC_Tank2. This is because it’s pretty hard to separate out live from dead larvae until the live larvae have grown up a bit. In case the dead larvae still in SS_Tank1 and HC_Tank1 cause additional mortality, I want to start a 2nd replicate where none of the larvae experienced the temperature drop.

• Checking for larvae in larval catch buckets
  • Maybe/Some larvae: SS1
  • Lots/Noticeable larvae: HC3, HC4, HC1, SS4
  • Also screened NF5 but did not get any larvae
  • Added to HC_Tank2 and SS_Tank2
• Counting larvae
  • need to get better at estimating ~100 per mL. Some of these counts had over 400 per mL which makes it harder to count and more stressful for the larvae when they’re in the tripour beaker

Larval counts data DNA samples

• As per my conversation with S. Roberts yesterday, I’m keeping all of the samples from my larvae counts, including those I don’t think are necessary for future genotyping. I’m trying 2 different preservation methods, and will do test DNA extractions next week to look at quantity and quality.
  • 75%/90% ethanol: this was suggested by Jake Heare. I pipet up one of the 1 mL larvae count samples and put it in a 1.5 mL tube. Since I used ethanol when counting, all of the larvae sink quickly to the bottom and I pipet out as much seawater as I can. I continue adding the other larvae count samples and decanting off the seawater in the same fashion. If needed I spin them down briefly with a centrifuge. Then I add 75% ethanol to the tube. A day or 2 after I take out the 75% ethanol and add 90% ethanol. These are stored in -20C.
  • RNALater: As described above, I take out as much seawater as I can and then add about .75 mL of RNALater to the tubes. These are stored in a -80C freezer.

In the afternoon, I stopped by the Roberts lab on the UWashington campus to find some bench space for the test DNA extractions and pick up a HOBO temperature logger to stick in one of my broodstock buckets so I can get a better idea of what they’re experiencing in case a temperature malfunction happens again.