Priming Protocols – mewing-notebook

Setup

color coding key (zip ties):

4 aquarium tanks setup with 2 buckets each (2 replicates for each species / treatment).

Aquaria filled to ~4 cm deep with FW (marked with line on tank)
1240 mL of water per white bucket (30 ppt seawater). Don’t put silos in yet (even empty silos) as it can affect bucket water heating
Heating bar and temp gauge in each – heating bar in broader aquarium setup and temp gauge in the bucket.
Set temp on heater manager as desired, wait approx ~1 hr for water to heat, check temp and confirm bucket water temp with gun or glass thermometer.
Want Rphil tank buckets to be at ~34, want cockle buckets at ~26.
- Initial trials and literature showed that Manila seed can tolerate ~38C before moralities occur and ~30C before moralities for the small cockle seed (2mm). As such, both priming temps are 4C below minimum mortality temp.
For poly(i:c) groups: add 930 uL of Poly(I:C) solution (1mg/ml concentration) to each marked poly treatment bucket just prior (~10 min) to pacing the silos in their treatment buckets.
Once at temp, can place silos in their respective treatment buckets.
- in treatment buckets for 2.5h – check temp one random silo (since all buckets are in same cold room / ie. exposed to exact same temp conditons) every 30 min w gun to confirm and record temp.

8 buckets setup on counter in tank room (no aquarium water bath needed)

1240 mL of water per white bucket (30 ppt seawater). Don’t put silos in yet
For poly(i:c) groups: add 930 uL of Poly(I:C) solution (1mg/ml concentration) to each marked poly treatment bucket just prior (~10 min) to pacing the silos in their treatment buckets.
Check and record temp prior to placing silos
Once at temp and warm temp experiment has started, can place silos in their respective treatment buckets.
- in treatment buckets for 2.5h – check temp every 30 min w gun to confirm and record silo temp

at the end of the priming duration, transfer silos to the yellow tanks (~10C) for 1 hr.
put poly(i:c) individuals in one of the yellow tanks post priming for ~30 minutes to allow them to flush the solution out, and then do a water change on that tank, and all other individuals in the other tank
- WQ is checked the morning of the experiment for both tanks to ensure recovery conditions are consistent across tanks
After recovery, put manilas and cockles in their normal tanks as per usual. closely observe over the next 48 hrs

^ June/July experimental logistics timeline

Code names:

MC - Manila clam

BC - basket cockles

letter = replicate group / aquarium

# = treatment silo within replicate group

Silo Counts:

MC-A1: 700

MC-A2: 700

MC-B1: 700

MC-B2: 700

MC-C1: 700

MC-C2: 700

MC-D1: 700

BC-A1: 203

BC-A2: 297

BC-B1: 300

BC-B2: 298

BC-C1: 315

BC-C2: 315

BC-D1: 315

BC-D2: 315

Tank setup:

Temp readings:

	T0 Initial	T1	T2	T3	T4	T5 (Final)
Time (PST)	12:38	13:06	13:37	14:09	14:40	14:06	Average
MC-A1	30.9	33.7	33.1	33.1	32.5	33.1	32.7
MC-A2	30.2	32.8	32.6	32.3	32.2	32.7	32.1
MC-B1	31.6	33.1	32.5	32.6	32.4	33.0	32.5
MC-B2	32.0	33.5	32.6	33.0	32.4	33.0	32.8
BC-C1	26.9	28.1	27.8	27.6	27.6	27.2	27.5
BC-C2	27.5	27.8	27.5	27.6	27.5	27.3	27.5
BC-D1	27.2	28.1	27.8	27.6	27.5	27.7	27.7
BC-D2	27.5	28.2	27.8	27.7	27.5	27.6	27.7
*note: cold temp was not recorded at same intervals for this week. 1 temp was recorded at T3 (halfway)	10.5

tank setup:

temp readings: