Shelly’s Notebook: Fri. Nov 9, 2018

Geoduck Broodstock experiment

Supplies for Pt. Whitney

  • -20C mini freezer: Steven’s going to try to pick one up this weekend, or I’ll order one next week if not in stores.
  • Ordered probe clips and hanging hooks from amazon to try to fix probes in tanks. I plan to mount the clips and hang the clip mount by zipties from the hanging hooks. We’ll see if it works.

Water chemistry

  • Apex and cameras have been offline since yesterday morning. Matt said their internet plan got canceled possibly because a bill didn’t get paid, but he will let us know when it’s back up.
  • We talked about setting up an independent modem to run the Apex systems on, and to also set the router up to auto-reset every day so we don’t have outages that could interrupt the treatment or data collection.
  • Here’s the Apex data from Oct. 26-Nov 7 and plots of the pH and temp over time.
    • it’s not convincing that the treatments still run when the internet is down so we will test this on Monday.

Reproductive Maturation Assay development

  • Grace shared some info about tissue samples for RNA extraction and qPCR
    • need to select samples based on development stage
    • need to design primers for VTG and b-actin
    • need to find the qPCR protocol and make sure we have all the reagents

Repeated hemolymph sampling: geoduck status @ SAFS

@12pm

  • Ducks look alive and OK IMG_20181109_120202.jpg

Transferred ducks to ~5L seawater IMG_20181109_120642.jpg

Added 5mL Shellfish diet and mixed with air stone IMG_20181109_120818.jpg

@2:30pm IMG_20181109_143024.jpg

@4pm IMG_20181109_160419.jpg

  • Feeding recommendations: Shellfish Diet feed per day (ml) = grams of broodstock meat (WET weight) x 0.036
    • probably could feed them a little more; ordered shellfish diet today

Shelly’s Notebook: Thurs. Nov 8, 2018

Geoduck Broodstock Experiment

Geoduck check-in and hemolymph sampling @ 3pm

 - both animals were still alive and OK ![](https://raw.githubusercontent.com/shellytrigg/P_generosa/master/img/IMG_20181108_151643.jpg) - one animal had a starfish attached to back end - one animal had got its syphon through the holes in the container and a crab and urchin were eating it - sampled "Star" one from wrong end, pulled out tea-colored fluid - stuck from correct end once, but at too steep an angle - stuck again at ~45 degree angle and in front of (anterior to) the hinge and collected about 1mL of hemolymph - sampled ["Chewy"](https://raw.githubusercontent.com/shellytrigg/P_generosa/master/img/IMG_20181108_152248.jpg) one time in the correct area and got 1mL hemolymph on first try  

– visualized hemolymph under 10x and 40x and saw clusters of cells, but not as many cells as last week (no pics) – From Michael: “We definitely see a ton of variability day to day in hemocyte numbers. And the amount we saw here in you geoducks would have been a huge amount for a normal Mya arenaria. I am attaching those two photos. They were done on a slide with a convex surface, so a plain flat slide with a coverslip will have less volume and fewer cells visible. Also, this was after about 5-10 minutes, as I recall, and if the cells are allowed to settle for 30 or so, then most of them should be attached and spreading out.”

 - spun cells at 1000xg for 5 min at RT (because 4C centrifuge kept throwing Err 3). It seemed like most of the cells pelleted, although some may have still been in the lymph. Separated lymph from cells and froze at -80 C. - returned animals to original container by 4:30pm with small holes to protect them, and added air stone to it.  

Lab meeting suggestions: