Start of full-scale larval OA experiment!

Wednesday, 2016-07-27

Today started like many of the previous days, with checking for extruded eggs and counting the larvae in some of the silos from the preliminary experiments. I had a late start at the hatchery because I was picking up a pipette gun from campus.

Extruded Eggs:

  • BC3T
    • 2 new: 112(2), 113(2)
    • 1 repeat: 34 (1)

Screened out silos at Day 5 from experiment started 2016-07-22 with OR2T and CA2T larvae. Took pictures under microscope at 10x of all but A84 and A83.

ID Family Sample Action
A86  OR2T 750 in RNALater Put in clean silo
A85  OR2T 750 in RNALater Put in clean silo
B61  CA2T 750 in RNALater Put in clean silo
B59  CA2T 750 in RNALater Put in clean silo
B65  OR2T 750 in RNALater Put in clean silo
B63 OR2T 750 in RNALater Put in clean silo
A84  CA2T 750 in RNALater Put in clean silo
A83  CA2T 750 in RNALater Put in clean silo

Then Sean and I started screening out the newly released larvae from the trays and buckets. There did not seem to be enough larvae from all three populations, so I stayed to finish screening out the last couple of buckets and count larvae. While the BC3T oysters were sitting in a bucket waiting to be placed into a clean tray, they started to release A LOT of larvae. Once I finished screening the last of the buckets I realized that we finally had enough from all three populations to start the experiment. Hooray! However, it meant I ended up staying until 10pm setting up by myself.

I had to remove some of the silos from preliminary experiments that were in the system in order to make room for the full experiment. I screened these out and left in tripours overnight to count the next morning.

  • B72, A63, A6, B66, B60, B55, A88, B66, B80, A100, A97, B51, A9

There were two family groups for CA and OR that had a lot of larvae, whereas BC had one group with a lot of larvae and one group with ~20,000. I decided to combine the BC groups, then have 2 replicates per treatment for each family group, so 4 silos per population/ treatment for CA and OR and 2 silos per population/treatment for BC. Not ideal, but I wasn’t sure when I would have at all three populations spawning at the same time again. I counted the larvae for each population with 4 subsamples of 0.5 mL, took the average to get larvae/mL and then calculated how many mL to add to each silo to get 15,000 larvae. I dispensed larvae with a pipette gun and 25mL pipettes while plunging constantly to mix, using a new pipette for each family group. When pipetting out replicates, I alternated between treatments to help mitigate possible effects of the plunging/pipetting process (probably a little overkill).

Family larvae/mL  mL added Experiment labels
OR1T  152.8 98.2 B20, B18, A72, A73
OR5B  153 98 A59, A71, B13, B11
CA4B  168 89.3 B19, A58, A57, B06
CA2T  290 51.7 A55, A56, B16, B12
BC3T+BC2T  232.5 64.5 A53, A54, B35, B45

I had hoped there would be enough larvae from BC1T, but there was only ~10,000.