Thur. Aug. 29, 2019 Sequim dig and Pt. Whitney Juvenile Exp. Setup

Sequim Dig

Kaitlyn and I took the 5:35am edmonds-kingston ferry to meet Liz and Steven at the Natural Resources Lab in Sequim Bay by 7:15am

Sequim Bay, from Natural Resources Lab

Oyster and geoduck plots in Sequim Bay. Boat is manning a stinger.

Remaining mesh tubes in our juvenile plot from 2018 (rows A-H columns 4-8)

met Liz at the Natural Resources Lab 861 Old Blyn Hwy Sequim, WA 98382
she supplied:
- 2x 5 gallon buckets with lids
- 2x trowels
- 1x clam gun (6” diameter?)
- 2x PVC tubes (12” x 10”)
- 2 pairs rubber gloves
we supplied:
- cooler with damp towel and ice packs
- mesh bags and color coded zip ties for the retrieved animals
  - blue = blue + yellow groups
  - red = red + purple groups
  - green = pink + green groups
  - orange = orange + gray groups
wide PVC pipe was meant to prevent the walls of the hole from caving in and getting flooded with water shown in picture below Kaitlyn and Liz digging

shimmy the wide PVC pipe down around a mesh tube
rock the mesh tube back and forth to pull it up
check core inside mesh tube for animals
- it helps to sift through the core on a flat surface that isn’t mud; we used the 5 gal. bucket lids for this
check hole for animals by feeling gently for siphon
if siphon felt, we’d keep one hand on the siphon and use the other hand to dig around
animals shells broke by trying to pull them up through the wet mud/sand so digging from underneath and all around helped prevent broken shells

Steven and Kaitlyn digging

In total we recovered:
- 26 live animals
- 1 dead animal (only shell)
- 14 crushed animals
- 15 locations had 0 animals
- data here under tab ‘8/29/2019’: Geoduck.dig.tracker
We came across a number of other puget sound clams (manila clams, cockles, macoma clams. Macomas appeared the most similar to geoducks. Here’s an example of macoma clam shells:
Overall, more successful than previous attempt but still could be better

a gentle stinger, Liz suggested something like a garden sprayer backpack
gloves: our fingers got cut up from digging, but feeling where the animals are is important so you don’t crush them. So I’m not sure how we would feel the animals if we had gloves on
a wide-diameter clam gun to fit around the mesh tubes; this way we could core the whole area hopefully without decapitating any. We could build this
longer mesh tubes that are closed at the bottom; this would ensure animals are in the same spot we planted them and we could account for natural mortality better

moved triploid broods from tote #4 to tote #6 with the other leftover broods
removed silos + spray bar containing manila clams from tote #4
in tote 4: placed each juvenile in a small piece of PVC (1” x 1/2”-1”) to stand upright (like the broods).
- Each PVC was color coded with zipties to match their group color
- groups were also kept separate on a mexican tray weighted down by a rock

Initial photos of animals before combining trays
- T3:
- T3 more upclose:
- T5:
- T7:
- T7 with ruler:
- T1:
Combined T3 and T5 var.low pH history trays and screened them on 5000um, 4000um, and 2350um mesh
- 5000um mesh:
- 4000um mesh:
- 2380um mesh:
- rinsed mesh contents into separate beakers
Did the same size selection and separation for ambient trays T1 and T7
5000um selection, top beaker (blue tape) = ambient progeny; bottom beaker = var.low pH progeny:
4000um selection: top beaker = ambient progeny; bottom beaker = var.low pH progeny:
2380um selection top beaker = var.low pH progeny; bottom beaker = ambient progeny:
almost all of the debris that came through 2380um mesh was empty shells from dead animals, so this worked really well for removing those

used same method Matt showed me before
for each parental history, I counted out equal amounts of animals from each size class and combined them in one master mix
5000um animals:
- ambient progeny visually had the least number of animals so counted those first
  - 182 animals
- for var.low pH animals, I counted out 182 animals and set these aside in tripour beakers in 15C (ambient) water bath
4000um animals:
- ambient progeny visually had the least number of animals so counted those
  - 462 animals in 77 mL (counted 78 animals in 13mL); added these to ambient master mix
- for var.low pH progeny, I measured out 77mL since these are the same size class; added these to var.low pH progeny master mix
2380um animals:
- var.low progeny visually had the least number of animals so counted those
  - in 10mL, 60-70% appeared to be dead/empty shells
    - 56 live animals in 160 total shells
  - total volume was 60mL, so 336 live animals; combined with var.low pH progeny master mix
- for ambient progengy, these also appeared 60-70% dead/empty, so I measured out 60mL and combined with ambient master mix

MASTER MIX SUMMARY

extra animals were returned to heath tray on H2 stack:
- amb tray: 2380-4000um (small size) only
- var.low pH tray: 4000um-5000um (medium size) + >5000um (large size) animals

while I was counting (with Kaitlyn’s assistance), Steven:
- drilled holes in all the waste bins beneath each silo so that silos still maintain 3” water
- repaired leaky threaded adapter port with plumbers putty
- swapped var.low pH with ambient water (including in waste bins) so both totes 2 and 3 manifolds are flowing ambient water from tote 5; this way we don’t have to start the treatment yet

For each parental history, distributed 980 animals across 10 replicate silos (5 silos for ambient and 5 silos for var.low pH chronic stress)
- ~ 98 animals/silo
- total master mix volume was 149mL; 149/10 ~= 15mL
- measured out 15mL and made piles that looked like 15mL
- transferred 15mL pile to mesh and gently placed mesh upside down in silo containing 3-4” water and 1/2” of sand

got L/S norprene 24 masterflex hosing flowing into the tote water supply plumbing; pump still set at 160

wrapped up the day around 6:30pm
var.low parental history had fewer animals but of larger size class
- this could be an effect of tray position during a critical developmental timepoint
Because the leftover var.low pH progeny were from different size classes (medium and large) than the left over ambient progeny (small), we decided to not outplant these
We decided against outplanting on Aug. 30 since we wouldn’t have time to make our outplanting optimizations outlined above and postponing outplanting would allow us to focus Aug. 30 on integrating the Sequim and hatchery 1.5yr old juveniles into the current juvenile experimental set up.