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.

Larvae collection, counting, and sampling

I’m being really paranoid at this point as I want to try and “catch ’em all”. I’ve modified my larvae collecting a little bit by stacking a 400 micron screen on top of the 100 micron screen to catch the poop/gunk.

• SS3, SS5, SS4, HC4, and HC5 had low or suspect number of larvae
• SS1, SS4, SS2, HC2 had a definitely noticeable or large number of larvae
• HC4 and HC5 did not have any larvae when checked with the dissecting scope
• Data sheet with larval counts

After I counted the larvae samples, I dumped them into into the appropriate HC or SS 100 L tanks, after they had been cleaned and filled up with water. In the afternoon, Ryan (the hatchery manager) told me that there had been a malfunction with the heated seawater line sometime between Friday afternoon and Saturday when he came in. All of the tanks were at ~15C, and they observed severe mortality in their larvae tanks. The larvae I had collected from the larval catch buckets were mostly healthy. There were a few empty shells (from now on I’ll count those too), but they were definitely less than 5%. The problem might be if there was a severe mortality event in my 100 L tanks. Those dead larvae would have been filtered out with the healthy ones while I was cleaning and put back in, this could make the rest of the larvae sick from swimming around with dead ones. Unfortunately I had already put my newly collected larvae in those same tanks.

There were 29,000 larvae in the HC tank and 87,850 in the SS tank over the weekend. There are now 83,600 larvae in the HC tank and 363,437 in the SS tank. On Wednesday when I clean these out, I will filter them over a 160 micron screen stacked on a 100 micron screen. I’ll do counts of both sizes, and in particular look at the proportion of dead larvae in the smaller size. Any larvae that didn’t die or get sick on Saturday would have grown since, while those that did would still be passing through a wider screen.