Separating host, holobiont, and symbiont fractions for metabolomics

THIS STEP MUST BE DONE PRIOR TO METABOLOMIC EXTRACTIONS!

This protocol details the separation of host, holobiont, and symbiont fractions from Montipora capitata larval samples. Fractions will be used for metabolite extraction. Protocols were developed by KW, AH, and JA.

• Stable isotope metabolomics larval protocol
• Stable isotope metabolomics extraction protocol
• AH and JA working protocol document

The samples used in this separation are from A. Huffmyer’s 2021 M. capitata larval experiment. For experimental details, see the tag “Mcapitata LarvalTemp 2021” in A. Huffmyer’s lab notebook.

Equipment & materials

• 1.5 mL tubes
• Homogenizer
• P1000 + tips
• 10% bleach
• 70% ethanol
• DI water
• Dry ice

Protocol

Before starting, label the desired tubes. The sample processing list is here. For each biological sample (indicated by #), label one of each of the following tubes:

1.5mL tubes:

• #-Host
• #-Sym
• #-Holo

Sample type separation order: C12 controls, C13 dark, C13 light

1. Thaw sample (2 mL screw cap tube w/ 1 mL of larvae) on dry ice.
2. Add 300 uL of ice cold DI water to sample.
3. Homogenize the sample for 1 minute on ice (or until larvae are completely homogenized). Use the homogenizer tip to get all the larvae from the sides of the tube.
4. Remove 150 uL of the homogenized holobiont and place into new tube (#-Holo).
   • Store on dry ice for further processing or put into -80°C.
5. Remove the remaining homogenate and place into new tube (#-Sym)
   • This will be around 150-250 uL of liquid. The volume will be variable because there may be excess seawater in the larval samples.
6. Spin the “Sym” tube at 9000g for 3 minutes at 4°C.
   • Make sure the homogenate in the “Sym” tube is liquid (as opposed to slushy) prior to centrifuging.
   • If supernatant is cloudy after centrifugation, spin again for 1 minute.
7. Remove the supernatant and place into new tube (#-Host)
   • This will be around 150-250 uL of liquid.
   • Store on dry ice for further processing or put into -80°C.
8. The “Sym” tube now only contains a symbiont pellet.
   • Add 150uL of DI water to the tube with the symbiont pellet. Either pipette up and down to mix or store on dry ice for further processing / put into -80°C.
   • The symbiont pellet is very dense and didn’t break up easily when pipetting up/down and vortexing. I added the DI water to the tubes and froze them without mixing. I will thaw and fully mix if we end up doing metabolite extractions on the symbiont fractions.
9. All sample fractions (Holo, Host, Sym) can be stored “Pre-Processing” freezer box in the -80C
10. Critical!!!! In between samples, clean the homogenizer to prevent cross-contamination
    • Wipe the motor with a kim wipe
    • Clean the motor (in this order) with DI water, 10% bleach, 70% ethanol, DI water
    • Wipe the motor with a kim wipe
    • Clean the motor in between samples and refresh all washes in between sample type

Prior to extractions, I might thaw and spin the host samples again to get rid of all debris.