Laura’s Notebook: Analysis of QuantSeq Larval Sizes

Check out my RMarkdown notebook where I analyze larval size upon release by parental pH and temperature treatment: laura-quantseq/notebooks/Larval-size-on-release.html

Here’s a sneak peak at one of the plots generated in this notebook:

larval-min-feret-mean.png?raw=true

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Shelly’s Notebook: Thur. Sept. 5, 2019 Pt. Whitney Juvenile Var.low pH experiment

Experiment Set-up

  • labeled silos
    • Tote #2 = 2.1-2.12 back to front and right to left
    • uc?export=view&id=1H3HV9m0oxPGV9dgEM79BAbTWBGwGZIyK
    • Tote #3 = 3.1-3.12 back to front and right to left)](https://ift.tt/1xJAnEZ
    • uc?export=view&id=1fS75J_o6hnsis9MrqNZ8dNv9XR4h0uaU

Animal status

  • ambient progeny appear less healthy than var.low progeny. It’s hard to tell how many are actually alive, but majority of the ambient progeny look dead. Many ambient and some var.low progeny have some growth that looks like what Matt saw earlier this season:
  • what Matt saw before: uc?export=view&id=1yEtg4b5RlgGtunx7g30XGFL51pzCSC2K
  • Silo with ambient progeny: uc?export=view&id=1eiXn0a0Ndvs3MvmZxBji8x4ES12LNzN8
  • took pictures in all silos to compare:
  • Silos with ambient progeny:
    • 2.1: uc?export=view&id=1Y1K93F5WAzgh0C0Z3elFXEJEF8pbAhRt
    • 2.2: uc?export=view&id=1tZNFEI03UVpYVYTW74GgOQ-Fu-O9cjqW
    • 2.3: uc?export=view&id=1obkbUYOJjDIf5KpGhhHmP0NawfnFBvvv
    • 2.4: uc?export=view&id=1x3XhUexEpOlDqAhTlTqhVlMWybsK0OJ5
    • 2.5: uc?export=view&id=1ysX7RZLlGIoSOZwdW9V-tSdTWH18bnda
    • 3.1: uc?export=view&id=1cLhh-qI65SwOWi0zq2sd8-gJ0fno5okV
    • 3.2: uc?export=view&id=11GER6CLuDqArdeRaL1nA86b7BpjvVR89
    • 3.3: uc?export=view&id=1DB95HCoH7Y6jD6kXTRCJWMCo5b_Nrs7g
    • 3.4: uc?export=view&id=1uSqzq4wMFcWEKxO9FrldczD4B0usvRPb
    • 3.5: uc?export=view&id=1vDcn7hVRtu1sNMMg2RN0Ed794vuL-uPA
  • Silos with var.low progeny:
    • 2.8: uc?export=view&id=1waC2Fq2eQqMC6IE0tIFgjHviTCOMm89m
    • 2.9: uc?export=view&id=1aVzIhyOzBabfMJxg3LA9okoBVnylKLJn
    • 2.10: uc?export=view&id=1oFmTMxltso74_eJugqYU5yDKMP9xtPod
    • 2.11: uc?export=view&id=14XB4bojWksMBE9OVScLB4GG-AcHk2GZd
    • 2.12: uc?export=view&id=1F2Pl1-nNKop8xsgZIsfQDHw0RNpKyLzF
    • 3.8: uc?export=view&id=17BRV9K9QFsj9ozM2qzNiXKUCOxLycXWu
    • 3.9: uc?export=view&id=1IgPeuCnp215R71pKx3JytSD02PWtWEk7
    • 3.10: uc?export=view&id=1xBJVzStvtTeeHl0MGXNaR2Xc9U6PWiqg
    • 3.11: uc?export=view&id=1GzErwBOfxf-wECemlicpMjJc7Hizvn9t
    • 3.12: uc?export=view&id=1vjPQMIxDdZHataGoxwGgpRI4svpiyFhT
  • Silos with 1.5year old juveniles:
    • 2.6: uc?export=view&id=1NrV6lheKy83y0DD9cMTrpWTouH1_Pn7x
    • 2.7: uc?export=view&id=1gPkB4eOskpkgVQzsSp6Tf9AWk18858ik
    • 3.6: uc?export=view&id=1bI6jwZgbpWmGE04glu7_jSUigtAIPViY
    • 3.7: uc?export=view&id=1QD3RtrGfUYwZ_EiEP6-7WZmn9yjh3z90
    • The macoma clams are more obvious now as their siphons are definitely different. Will need to go through and remove these, then count how many animals are left from each group

Water chem

  • took discrete measurement on low pH tank to confirm Apex probe is reading correctly
    • discrete pH probe may be dying because it takes forever to stabilize. This only occurs around pH 7. It does not seem to take forever during calibration, however after calibration when reading the pH 7.0 standard it still takes a while.

EXP. START: SWITCHED TOTE 2 TO VAR.LOW SEAWATER AT 2PM

Respirometry

  • This required a couple hours of troubleshooting before any real trial could be started. Thanks to Sam for talking me through a lot of it!
  • software login is usrnm: admin pswd: admin
  • Needed to select com port 3 in order for the SDRv4.0.0 software to recognize the SDR plate
  • I used SDR vials that Sam most recently used: uc?export=view&id=1P5DgdS3XA7CYssE0mgwD12_yxHsdZ3yE
  • Sampled 3 animals/silo and held them in the little square tupperware containers (Sam previously used) in a water bath
  • Took pictures for normalizing by size
    • Tote 2: uc?export=view&id=1JzRQQJswaebGJgnvyBPbQk0WC5Q8Aq80
      • labels of tupperware: uc?export=view&id=1j9sKTz5FtvkuTIjaOXR44cfG-RX2Dl__
    • Tote 3: uc?export=view&id=12iNE-0cuekHd7PPzlh4p0AiP6lcsLY2d
  • Filling vials
    • took about 30 minutes:
    • Filled all vials with same filtered sea water
    • Used transfer pipettes as chopsticks to get animals into vials
    • vials for silos 3.5 and 3.12 contained only 2 live animals (I didn’t initially realize the third animals I had grabbed were dead)
    • took a picture of the plate orientation (A1 in the top left corner): uc?export=view&id=1T6az5W1MPb0Ou_TN_xwk3Lh0BDbgpUQE
  • taped plate to rotator as Sam previously showed me, moved rotator into incubator and switched on. THE BASE CAME OFF! The screw holes were stripped :
    • uc?export=view&id=1FfMB1xZjPm6pPXgb05tsm2wemQxqDmac
    • uc?export=view&id=1Oz43YxXqbkixqh4OJPJ47FXh5Fz5Lu45
      • took 15 minutes to repair with new, longer screws
  • Started running trial around 3pm and O2 levels were all over the place
  • uc?export=view&id=17r67MQBSk6_dSyNNpJShRMLFlJIc6WF7
    • Sam suggested it was likely from air bubbles and to remove 1mL from each vial and refill with fresh filtered seawater. I did this and results looked more normal but some still showed low values
    • uc?export=view&id=1SfdZBzqooM_wHhB09JRbYGLlUshZ02UF
    • Decided to just put 1 animal/vial to avoid hypoxic conditions

      1 animal/vial

  • i returned all animals back to their corresponding small tupperware containers in the water bath
  • I selected 1 animal from each container and left the remaining animals
  • size photos and plate orientation here
  • data table here
  • between run 1 and 2, i changed the water in the water bath and tuperware containers

sampling after each trial

  • after each trial, I got the wet weight of each individual:
    • placed it on paper towel to remove excess seawater
    • weighed it in a labelled microfuge tube on the balance
    • after recording the wet weight, I froze the sample @ -80C.

conclusions

  • I may have be causing acute stress to animals at different times during the respirometry trial:
    • hypoxic conditions during first attempt, albeit this was brief (less than 1 hour)
    • water bath at 16C left at RT (in AC) so may have increased in temp to 18-20C; I didn’t monitor this
    • by the time I froze them, they may have been eliciting a response specific to
      • being dried off
      • dropped into the microfuge tube
      • being dropped on the scale by accident (probably happened 5 times, but didn’t break the animal)
      • I’m hoping their parental history will override any short term response to unintentional stress

moving forward

  • tomorrow I was thinking about doing a respirometry trial, but maybe only 1 round since we are short on animals
    • pros: animals are still alive, and we could see a short term response
    • cons: it’s too short a time for a response?, killing more animals before they can get stronger

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