BIG UPDATE! Marrowstone has been incredibly busy with lots of experiments and lots of day-to-day tasks to keep on top of, that I put off posting an update… so here’s a big one.
Day 18 –> 2023-08-08
Morning:
MUSP
MUSP controls all look great.
MUSP exposed:
Bin1 –> Dermasterias looks fine; Pisaster dead; Pycnopodia has dropped an arm
Bin2 –> Dermasterias fine; Pisaster dead
Bin3 –> Dermasterias and Pisaster looks fine; at sampling time, the Pycnopodia had 1 arm twisted and 4 arms crossed
Bin4 –> Dermasterias and Pisaster look ok; Pycnopodia dead
Bin5 –> Dermasterias ok; Pisaster one arm crossed and remaining arms bunched; Pycnopodia dead
Bin6 –> Dermasterias, Pisaster, wild Pycnopodia, and Friday Harbor Pycnopodia all ok
Bin7 –> Dermasterias and Pisaster ok; Pycnopodia has one arm curled (though next to the FH Pycnopodia) and at sampling time, wild Pycnopodia had 2 arms folded over body and a weird inflated thing at madreporite
Bin8 –> Dermasterias and Pisaster ok, Pycnopodia dead
We sampled coelomocytes from 12:44pm-3:47pm.
WAT (Water Transmission experiment)
Did some more pre-filterting and filtering of Day 0 samples.
Evening:
MUSP
Similar to the morning check.
WAT
Adult Pycnopodia A is a little twisty-looking, so we might be able to start the Water Transmission (WAT) exposures and officially start the experiment tomorrow!
Day 19 –> 2023-08-09
Morning:
MUSP
Controls –> all good!
Exposed:
Bin1 –> Dermasterias ok; Pisaster and wild Pycnopodia dead
Bin2 –> Dermasterias ok
Bin3 –> Dermasterias ok; Pisaster has 1 arm twisted; Pycnopodia twisty on tubes
Bin4 –> Dermasterias and Pisaster ok; Pycnopodia dead
Bin5 –> Dermasterias ok; Pisaster very twisty
Bin6 –> all ok
Bin7 –> all ok
Bin8 –> Dermasterias and Pisaster ok
WAT
Finished Day 0 TFF filtering with the final 3 juvenile samples
START WAT!!!!
Sample adults and juveniles coelomic fluid (I take coelomocytes for adults since I don’t have coelomocytes from adults that have been injected with sick CF for transmission –> I only have coelomocytes/RNA from adults that have bee n injected with raw homogenate from summer 2021 and summer 2022 experiments).
MUSP Evening:
Controls –> all ok
Exposed –>
Bin1 –> Dermasterias ok
Bin2 –> Dermasterias ok
Bin3 –> Dermasterias ok; Pisaster and Pycnopodia a little twisty
Bin4 –> Dermasterias and Pisaster ok
Bin5 –> Dermasterias ok; Pisaster very twisty - 4 arms twisted!
Bin6 –> Dermasterias, Pisaster, and Friday Harbor Pycnopodia all look ok; but the wild Pycnopodia is twisty!!!
Bin7 –> all ok
Bin8 –> Dermasterias ok; Pisaster 1 arm twisted and 1 lesion
Thoughts about Bin 6. At first I was worried about contamination since the wild Pycnopodia is sick and no one else is… but that doesn’t make sense. If there was contamination, I would have expected the FH pycno to get sick… so it could be some resistance happening? The FH Pycno that we injected with sick CF maybe never got sick but perhaps was able to still transmit?? To be investigated ….
Day 20 –> 2023-08-10
MUSP
FH Stars in exposed bins 6 and 7 still look fine, though the wild pycnos look a little bit like they might show signs. All controls look fine.
WAT
TFF filter some controls –> 1 L of autoclaved R0; 1L of tap water; 1 L of RO water (not autoclaved). Took 0.8um and 0.22um filters, and the viral concentrate.
Day 21 –> 2023-08-11
MUSP Morning
Controls all good
Exposed:
B1 –> derm ok
B2 –> derm ok
B3 –> derm ok; pisaster twisted; pycno dropped 5 arms
B4 –> derm ok; pisaster lesion
B5 –> derm ok; pisaster in a ball twisted
B6 –> derm ok; pisaster ok; wild pycno dropped an arm, low turgor, and twisted; FH pycno ok
B7 –> derm ok; pisaster ok; wild pycno ok; FH pycno ok
B8 –> derm ok; pisaster 3 arms twisted
WAT morning
All controls and all exposed look fine
PRES 2 Morning
All look good
MUSP sampling
sampled coelomocytes and coelomocyte supernatant for all remaining stars. MP sampled controls, I sampled exposed. We sampled the FH stars in bins 6 and 7, and control pairs in bins 14 and 15.
Some exposed samplings weren’t possible… reflected in our sample log.
MUSP evening
Controls all ok
Exposed:
B1 –> derm ok
B2 –> derm ok
B3 –> derm ok, pisaster a couple of arm twist; pycno arm drops
B4 –> derm ok; pisaster ok
B5 –> derm ok, pisaster has all 5 arms twisted
B6 –> derm ok; pisaster ok; wild pycno dropped more arms and about 5 arms twisted; FH pycno ok
B7 –> all ok
B8 –> derm ok; pisaster 2 arms twist
WAT Evening
all controls and expoesd ok
PRES 2
all ok
Day 22 –> 2023-08-12
MUSP morning
Controls all look good
Exposed
B1 –> derm ok
B2 –> derm ok
B3 –> derm ok, pisaster 2-3 arm twist, pycno dead
B4 –> derm ok, pisaster ok
B5 –> derm ok, pisaster all 5 arms twisted
B6 –> derm ok, pisaster ok, wild pycno only has one arm attached to disc (disc is still attached to side of bin, so not calling it dead yet), FH pycno ok
B7 –> all stars look ok
B8 –> derm ok, pisaster 2-3 arm twist
WAT morning
controls and exposed all good
Pres 2
all good
Co-housed disease line stars
Karen (adult pycno K) looks fine
Pycno A –> totally fine except 4-5 arm twist and juvenile pycno 30 that’s cohoused with A also looks ok.
Musp Evening
controls all good
Exposed
B1 –> derm ok
B2 –> derm ok
B3 –> derm ok; pisaster a little twisty
B4 –> derm and pisaster ok
B5 –> derm ok; pisaster twisty
B6 –> derm and pisaster ok; wild pycno dead; FH pycno ok
B7 –> all ok
B8 –> derm ok; pisaster twisty
WAT Evening
controls and exposed look good
PREs 2
All look ok
Day 23 –> 2023-08-13
MUSP
No new updates… things look same as yesterday.
WAT and PRES 2
No new updates…
Day 24 –> 2023-08-14
MUSP Morning
Controls all good.
Exposed no new updates… same as previous 2 days
WAT morning
Adult 2_J_ERCF twisty, so sampled water with control pair (2_I_HKCF). Also sampled CF (n=3 with 750ul shield, n=3 just frozen) and did 2 smears per adult.
Started TFF at ~1pm.
MUSP
Sampled MUSP.
MUSP Evening
No new updates.
WAT Evening
No new updates… but TFF finished around 10:30pm
Day 25 –> 2023-08-15
MUSP morning
Controls –> all ok
Exposed:
B1 –> derm ok
B2 –> derm ok
B3 –> derm ok; pisaster puffy and twisted
B4 –> derm ok; pisaster crossed/twisty
B5 –> derm ok; pisaster looks puffy and crossed
B6 –> derm, pisaster, and FH pycno look ok
B7 –> all ok
B8 –> derm ok; pisaster low turgor
WAT
TFF for adult pair 3
REX START
There were 6 stars that survived their initial exposures from different experiments, so we re-exposed them with 200ul of ERCF.
1_19_REX (previously 11_19_50ERCF from TIT)
2_18_REX (previously 10_18_100ERCF from TIT)
3_28_REX (previously 18_28_150ERCF from TIT)
4_3_REX (previously 5_3_ERCF from FHW)
5_56_REX (previously 1_56_ERCF from FHW)
6_21_REX (previously 3_21_RERCF from PRES 1)
Day 26 –> 2023-08-16
MUSP morning
All controls ok.
Exposed… not sure –> I was packing up things to bring to folks at USGS.
I helped sampled a little from WAT so that we could bring CF from Karen and the control adult pycno to John Hansen at USGS Seattle who will be characterizing coelomocytes.
I also gathered up some smears for Aine Hawthorn.
I also brought boxes of PSC (coelomocytes in 300ul DNA/RNA Shield) and PSCS (coelomocyte supernatant straight frozen) to Roberts Lab at UW and put in -80.