date created: 20210625; date last revised: 20210625 hgr post creation

Lab notebook post recapping AT RB onboarding to polyp bailout experiment and plans moving forward

1. Corallite counting

• protocol here; data in private repo
• changes to protocol: N/A
• remarks on protocol onboarding:
  • protocol best done with extra light shining on skeleton
  • for onboarding purposes, 2 people would count each skeleton (data in lab notebook, counters were within 5% of one another but sometimes larger differences ~9% occurred when counting larger fragments)
  • when two people count the same fragment, have the first person mark each corallite with a lighter-colored sharpie so the second person can still track the corallites they count with a darker-colored sharpie
  • thin sharpies work best
  • observed a consistent relationship between # corallites and # micropropagules

2. Micropropagule (MP) counting

• protocol here; data in private repo
• changes to protocol: updates to bailout portion of the protocol described here are optimized fo incubator setup
• remarks on protocol onboarding
  • protocol best done under light
  • was able to distinguish micropropagules (darker round clumps of tissue that sometimes had “mouth” “donut” holes in the middle) from mesenterial filaments (lighter scraps of tissue) without the microscope. mesenterial filaments were NOT counted towards the total micropropagules released because the are scraps of connecting tissue rather than a bailed out polyp
  • for training, 3 people would count the MP’s released by each fragment. The first person would count them from the mesh chamber into a different cup (with seawater), the second person would count them into the mesh chamber, and the third person would count them back into the the cup with seawater
  • MPs were counted by eye with a bulb pipette. It is best to count 3-5 MPs at a time as they are drawn into the bulb pipette
  • All counters were careful not to let MPs get stuck in the bulb pipette
  • counting of MPs was consistent among counters and had a strong relationship with # corallites on the skeleton
  • the final MP count corresponded to the number settled into the 6 well plates.
  • recording the counts by settling into well plates helped maintain focus. This is because we were focused on counting to intervals of 20 (for each well + any extra after reaching the last multiple of 20) which prevented distraction and miscounting
  • moving forward will count MPs when settling into well plates

3. Settlement, water changes, and feeding of micropropagules in 6well plates

• protocol here, needs to be updated; data in private repo, abbreviations described in readME
• changes to protocol:
  • folding over corner of waterproof paper made for easier removal from well
  • microscope coverslips are very difficult to remove from well, need to add a tape flag on side
  • want to order small 1in coral disks to use as settlement substrate
  • water changes: (weekdays, did not evaporate over weekend) included removing 3-4mL of seawater with a bulb pipette and adding 4-5mL of 0.22uM filtered seawater with a bulb pipette. At no point were the MPs exposed to air. Bubbled in oxygen to source container of filtered seawater before pipetting
  • feeding: weekly feeding on fridays. Added reef roids to a 50mL falcon tube of filtered sw and inverted tube to mix food. 1mL of was pipetted into each well. Plates were placed back in the incubator for 1 hour. To replace the water after feeding, the settlement substrate (one ate a time) was temporarily placed in a different container with filtered seawater. All ~10mL of water was removed from the well and ~10mL of filtered seawater was added to the well. The settlement substrate was then placed back into the well.
• remarks on settlement checks, more in subsection 4
  • checking and characterizing each potential settlement event under the microscope took 2-3 hours (for 6 well plates). Will want to schedule strategicially (1-2x/week)
  • compound scope much better for settlement checks than dissecting scope
  • counting # of potential settlement “events” by eye looking at the settlement substrate much more feasible as 3-4x/week task

4. Remarks on settlement tracking

• location of data in private repo, abbreviations described in readME
• MPs “attached” (loosely, can be uprooted by pipetting up and down) to waterproof paper within 24hr.
• A small fraction of MPs were in the well and appeared to be degenerated polyps. Unfortunately, there was a MUCH lower frequency of recovered polyps than when settlement chambers were placed in the water tables
• Many of the MPs “attached” to the waterproof paper wound up being piles of disintegrated tissue (no clear body patterns, looked like blobs of symbionts, didn’t have enough “height” that required refocusing compound microscope)
• Other MPs “attached” to the waterproof paper didn’t have clear body patterns but had some semblance of morphology (needed to refocus microscope to examine vertical profile of micropropagule). Will continue to monitor these

5. Refining the salinity ramp

• The salinity ramp has yet to “work as expected” in the incubator (first trial didn’t program pump correctly, second trial resulted in overdose), details in 20210622 lab notebook post here
• 48 doses of ~79psu seawater needed to complete salinity ramp. Timed from monday evening - wednesday 20210120_micropropagule_phenotyping
• length of salinity ramp might cause extra stress on Pocillopora and lessen likelihood they will have energy for settlement.
• 20210628 trial - start monday morning, stick to 25mL doses. can reprogram pump at end of day (only allowed to schedule 24 doses/day) so the 48 doses can take place between ~11am monday-~11am tuesday. If bailout does not occur by 11am tuesday will be able to monitor for rest of working day. Could also start with 1000mL instead of 1100-1200mL in mini tank so the salinity increases at a more rapid rate.