Isolation of Symbiodiniaceae from scleractinian coral hosts suitable for cryopreservation and laser-warming

Select 3-5 genotypes for each target species to ensure a sample representative of the Symbiodiniaceae commonly found in that coral species.

Take a photosynthetic yield measurement of each genotype using a PAM fluorometer to use as reference control value throughout the Symbiodiniaceae isolation protocol.

Blast coral tissue containing algal endosymbionts from the skeleton of each coral by using a dental flosser filled with FSW and collect the blastate (coral tissue and symbionts suspended in FSW) in a bowl or Ziploc bag. If isolating symbionts from a mushroom coral (e.g., Lobactis scutaria ) stay away from the mouth of the polyp to minimise mucus in the blastate and to allow for recovery of the fungiid. Aim for 250-900 mL of total blastate.

Extra step for corals with brittle skeletons, such as some Pocillopora and Acropora species: filter the blastate through a 200-µm filter to exclude any skeleton shards. In step 8, pay attention to any remaining skeleton shards. See the Guidelines, Troubleshooting section for further steps to remove skeleton shards. Step 9 can be skipped.

Pour the blastate, including the mucus, in 50-mL falcon tubes. Balance the tubes so that they have similar volumes of liquid and mucus using a transfer pipette.

Spin the samples in their 50-mL tubes, in a centrifuge for 10 min at RCF = 2300 x g.

For each 50-mL tube: transfer the floating raft (above the supernatant) into a 15-mL glass tissue grinder, discard the supernatant, and resuspend the pellet in 1-2 mL FSW using a 1-mL pipette. Add the resuspended pellet to the floating raft in the glass tissue grinder.

Add some FSW to the sample and homogenise the contents of the glass tissue grinder using the loose pestle until the solution appears homogeneous. This may take some time for coral species that naturally harbour a lot of mucus such as Lobactis and Porites species, and may be faster for other species such as Acropora and Pocillopora species (pers. obs.)

Transfer the homogenised solution through a 200-µm filter and into a glass beaker.

Repeat steps 7-9 for each 50-mL tube.

Transfer the filtered solution into clean 50-mL falcon tubes (aim for maximum 6 tubes).

Take a photosynthetic yield measurement in one of the falcon tubes using a PAM fluorometer, and record the value to ensure the symbionts are still healthy and yielding photosynthetic values near control levels.

Spin the 50-mL tubes in a centrifuge for 10 min at RCF = 2300 x g.

For each 50-mL tube: discard the supernatant and resuspend the pellet in 1-2 mL FSW using a 1-mL pipette. Transfer all resuspended pellets to a clean glass tissue grinder.

Add some FSW to the sample if needed and homogenise the contents of the glass tissue grinder using the loose pestle until the solution appears homogenous.

Transfer the homogenised solution through a 100-µm filter and into a clean 50-mL tube (aim for 1-2 tubes).

Take a photosynthetic yield measurement using a Junior PAM fluorometer, and record the value.

Spin the 50-mL tube(s) in a centrifuge for 10 min at RCF = 2300 x g.

For each 50-mL tube: discard the supernatant and resuspend the pellet in 1-2 mL FSW using a 1-mL pipette. Transfer all resuspended pellets to a clean glass tissue grinder.

Add some FSW to the sample if needed and homogenise the contents of the glass tissue grinder using the loose pestle until the solution appears homogenous.

Transfer the homogenised solution through a 70-µm filter and into a clean 50-mL tube (aim for 1 tube).

Take a photosynthetic yield measurement using a Junior PAM fluorometer, and record the value.

Spin the 50-mL tube in a centrifuge for 10 min at RCF = 2300 x g.

For each 50-mL tube: discard the supernatant and resuspend the pellet in 1-2 mL FSW using a 1-mL pipette. Transfer all resuspended pellets to a clean glass tissue grinder.

Add some FSW to the sample if needed and homogenise the contents of the glass tissue grinder using the loose pestle until the solution appears homogenous.

Transfer the homogenised solution through a 40-µm filter and into a clean 50-mL tube (aim for 1 tube).

Take a photosynthetic yield measurement using a Junior PAM fluorometer, and record the value.

Spin the 50-mL tube in a centrifuge for 10 min at RCF = 2300 x g.

For each 50-mL tube: discard the supernatant and resuspend the pellet in 1-2 mL FSW using a 1-mL pipette. Transfer all resuspended pellets to a clean glass tissue grinder.

Add some FSW to the sample if needed and homogenise the contents of the glass tissue grinder using the loose pestle until the solution appears homogenous.

Transfer the homogenised solution through a 20-µm filter and into a clean 50-mL tube (aim for a final volume of 50 mL / 1 tube).

Take a photosynthetic yield measurement using a Junior PAM fluorometer, and record the value.

Prepare an Erlenmeyer flask with 50 mL of double-strength Guillard’s F2 Marine Enrichment Solution containing double-strength Polne-Fuller antibiotic cocktail.

Gently add the Symbiodiniaceae solution to the Erlenmeyer while swirling the Erlenmeyer. The total volume should add up to 100 mL.

Close the opening of the Erlenmeyer flask with a filter paper or a circle of fine mesh fabric and tie it down with a rubber band.

Place overnight in an incubator at 24-26°C with a day/night light cycle.

The next morning, resuspend the Symbiodiniaceae solution using a 1-mL or a transfer pipette, and pour into 2-3 50-mL falcon tubes.

Spin the 50-mL tubes in a centrifuge for 10 min at RCF = 2300 x g.

For each 50-mL tube: discard the supernatant and resuspend the pellet in 1-2 mL FSW using a 1-mL pipette. Transfer all resuspended pellets to a clean glass tissue grinder.

Add some FSW to the sample if needed and homogenise the contents of the glass tissue grinder using the loose pestle until the solution appears homogenous.

Transfer the homogenised solution through a 20-µm filter and into a clean 50-mL tube (aim for max 50 mL / 1 tube).

Take a photosynthetic yield measurement using a Junior PAM fluorometer, and record the value.

Your Symbiodiniaceae solution is now ready. Use immediately for experiments, dilute to 50 mL in FSW if to be used later in the day, or dilute to 100 mL in an Erlenmeyer flask in Guillard’s F2 Marine Enrichment Solution if to be used on a later day.

Depending on the Symbiodiniaceae community composition, the solution will remain stable for at least 2-3 days.