Spawn night protocol

For embryonic development of Montipora capitata rice corals

Author

Sarah Tanja

Published

March 26, 2024

Prep

Make Stock Leachate

First make 400mL of each stock solution from the 1000 mg/L prepared leachate

  • Make 10 mg/L stock:

    1000 mg/L * V1 = 10 mg/L * 400mL

    V1 = 4mL

    400mL of 10mg/L stock = 4mL of 1000mg/L stock + 396mL of FSW

  • Make 1mg/L stock:

    10mg/L * V1 = 1mg/L * 400mL

    V1 = 40mL

    400mL of 1mg/L stock = 40mL of 10mg/L stock + 360mL of FSW

  • Make 0.1 mg/L stock:

    1mg/L * V1 = 0.1mg/L * 400mL

    V1 = 40mL

    400mL of 0.1mg/L stock = 40mL of 1mg/L stock + 360mL of FSW

  1. Then dilute the stock to each treatment vial/jar

For a 20mL scintillation vial for coral embryo experiments with a final target volume of 15mL:

1mg/L HIGH treatment

10mg/L * V1 = 1mg/L * 19mL

V1 = 1.9mL

19mL of 1mg/L leachate = 17.1mL of FSW + 1.9mL of 10mg/L stock

0.1 mg/L MID treatment

1mg/L * V1 = 0.1mg/L * 19mL

V1 = 1.9mL

19mL of 0.1mg/L leachate = 17.1mL of FSW + 1.9mL of 1mg/L stock

0.01 mg/L LOW treatment

0.1mg/L * V1 = 0.01mg/L * 19mL

V1 = 1.9mL

19mL of 0.01mg/L leachate = 17.1mL of FSW + 1.9mL of 0.1mg/L stock

ZFIX

Going from 18.5% to 4% in a ~200mL final volume!

We want a total of 200 mL volume

0.185 * V1 = 0.04 * 200mL

V1 = 43.24mL

To get 200mL of 4% ZFIX: Add 43.24 mL of 18.5% ZFIX to 156.76mL of millipore water

Freeze (& shield)

To preserve embryos for RNA extraction and sequencing we will first submerge them in Zymo DNA/RNA Shield, and then pronptly transfer them to a -80C freezer. Here, we won’t be dunking them in LN2. The combined DNA/RNA Shield and the quick freeze (due to small size) they will experience in the -80 are together sufficient for preserving RNA.

We will first very gently use a tip-clipped transfer pipette to transfer embryos to our 0.5mL screw-cap cryo-tubes. Wewill then carefully decant any excess FSW out of the tube, and then add 500uL of Zymo RNA/DNA Shield. Gently invert cryo-tubes a few times to ensure embryos are fully submerged. Once cryo-tubes are filled with their samples, arrange them in wax paper boxes and promptly shift them to -80°C freezer for storage until samples are ready to be processed.

Spawn Night

19:00 - 19:30

  • Move each colony to a numbered/named cambro chamber bin or 5 gallon bucket
  • Ensure each colony has enough headspace so that bundles can rise to the surface
  • Reduce blue holding tank water level to below the lip of the chambers
  • Decant water in chambers such that bouyant bundles don’t spill out

19:30 - 20:00

  • Organize the following collection materials at the spawning tubs:

    • 50mL falcon tubes (1 for ea. colony) filled with 30mL of 0.22micron FSW
    • Tip-clipped transfer pipettes
    • Red Headlamps (new batteries)
    • Printed waterproof spawn collection data sheet

  • Organize the following collection materials at the dry lab bench:

    • 20mL scintillation vials prepped with treatments

    • 50mL falcon tube rack (for working with active bundle-bundle crosses)

    • Tip-clipped transfer pipettes (for moving bundles to scintillation vials)

    • Printed waterproof bundle-bundle cross metadata sheet (for recording parent colonies of crosses)

20:50 - 21:20

  • Observe for setting (polyp expansion and bundle visibility) via red headlamp
  • Find a spawner!
  • Collect 24 bundles from each spawning colony and transfer them to a pre-labeled 50mL falcon tube with 30mL of FSW
  • Once we have at least 2 colonies worth of bundles, 1 person will move with any filled falcon tubes to the dry lab bench and begin bundle-bundle crosses
  • 1 person will remain and continue to collect bundles from colonies, periodically transferring them to the bundle-bundle crossing station at the dry lab bench

21:20 - 22:00

  • Cross fertilize by taking one bundle each from two distinct colonies and transferring them to the same scintillation vial
  • Record the parent colonies of each cross on the embryonic development metadata sheet
  • Only transfer intact bundles! Once eggs break up and begin to hydrate, the spawn party is over

22:00 - 22:30

  • Observe scintillation vials for bundle breakup and egg hydration

  • Record the times when the first and last vials experience hydration

    • First Egg Hydration Time_______________

    • Last Egg Hydration Time_______________

  • Average the times above , and add 4 hours, 9 hours, and 14 hours

    • Average Egg Hydration Time______________

    • 4 hours post-fertilization1______________

    • 9 hours post-fertilization______________

    • 14 hours post fertilization______________

  • Set alarms for ~40 minutes prior to the times above

22:30 - 23:00

  • Return each colony to blue tank and remove chambers

  • Return water height to normal

  • Clean up anything left outside

23:00 PM - 01:45

  • 2.5 hr nap time!

02:15 - 03:30

  • ZFIX 4hpf samples (4Z)

    • Use a transfer pipette to gently move embryos from scintillation vials to microcentrifuge tubes
    • Decant any excess FSW with transfer pipette
    • Add ~1mL of 4% ZFIX to each microcentrifuge tube in the fume hood!
    • Gently invert each capped scintillation vial to mix the ZFIX into the filtered seawater and sample
    • Record time of fixin’ : __________
    • Add 8-12 hours to fixin’ time to transfer from ZFIX to 70% ethanol: ____________
    • Set fixed vials in 4C fridge

  • Freeze 4hpf samples (4F)

    • Gently transfer live embryos to cryo-vials

    • Decant excess FSW using a transfer pipette

    • Add 500uL of Zymo DNA/RNA Shield

    • Promptly transfer to -80C freezer

  • Make PVC Leachate (on July 6th!)

03:30 - 06:45

  • 3.25 hr nap time!

07:15 - 08:30

  • ZFIX 9hpf samples (9Z)

    • Use a transfer pipette to gently move embryos from scintillation vials to microcentrifuge tubes
    • Decant any excess FSW with transfer pipette
    • Add ~1mL of 4% ZFIX to each microcentrifuge tube in the fume hood!
    • Gently invert each capped scintillation vial to mix the ZFIX into the filtered seawater and sample
    • Record time of fixin’ : __________
    • Add 8-12 hours to fixin’ time to transfer from ZFIX to 70% ethanol: ____________
    • Set fixed vials in 4C fridge

  • Freeze 9hpf samples (9F)

    • Gently transfer live embryos to cryo-vials

    • Decant excess FSW using a transfer pipette

    • Add 500uL of Zymo DNA/RNA Shield

    • Promptly transfer to -80C freezer

08:30 - 11:45

  • 3.25 hr nap time!

12:15 - 13:30

  • ZFIX 14hpf samples (14Z)

    • Use a transfer pipette to gently move embryos from scintillation vials to microcentrifuge tubes
    • Decant any excess FSW with transfer pipette
    • Add ~1mL of 4% ZFIX to each microcentrifuge tube in the fume hood!
    • Gently invert each capped scintillation vial to mix the ZFIX into the filtered seawater and sample
    • Record time of fixin’ : __________
    • Add 8-12 hours to fixin’ time to transfer from ZFIX to 70% ethanol: ____________
    • Set fixed vials in 4C fridge

  • Freeze 14hpf samples (14F)

    • Gently transfer live embryos to cryo-vials

    • Decant excess FSW using a transfer pipette

    • Add 500uL of Zymo DNA/RNA Shield

    • Promptly transfer to -80C freezer

Ethanol Transfer Times

4hpf: ______

9hpf:______

14hpf:______

Tip

You can do this!

Footnotes

    • (& on July 6th make another batch of leachate!)
    ↩︎