These instructions allow to quantitatively assess coral physiological parameters, i.e. tissue biomass (protein concentration), algal density and chlorophyll concentration.

Procedure

1. Remove the tissue from a frozen sample using an air brush within a 50ml falcon tube (if the sample was stored at -80°C the tissue will easily come off just by blasting air with the airbrush), then add 2ml of PBS/FSW to the tissue. If working with coral planulae or spats, add only 1ml of PBS/FSW.

2. Dry the skeleton and save it for surface area measure (aluminum foil method).

3. Homogenize the tissue with an electrical homogenizer.

4. Remove 1ml of the tissue to a 1.5 ml Eppendorf tube, keep the rest in the falcon tube as spare. If working with coral planulae or spats, remove 0.5 ml of the tissue to a 1.5 ml Eppendorf tube.

5. Centrifuge the 1.5 tube for 5 minutes at 5000rpm at 4°C.

6. Remove 100ul from the supernatant to a 0.5 ml Eppendorf tube for host protein measure. For protein concentration measurements I used the Micro BCA Protein Assay kit following the manufacturer’s protocol.

7. Remove the supernatant (host tissue) work with pellet (algae).

8. Resuspend the algae with 1ml FSW/PBS and homogenize.

9. Centrifuge for 5 minutes at 5000rpm at 4°C.

10. Remove the supernatant. If working with few amounts of coral planulae/spats (<10 per tube), go directly to step n° 14.

11. Resuspend the algae with 1ml FSW/PBS and homogenize.

12. Centrifuge for 5 minutes at 5000rpm at 4°C.

13. Remove the supernatant thoroughly

14. Resuspend the algae with 1ml FSW/PBS and homogenize.

15. Take 30ul for algae count, using an hemocytometer. See the protocol for counting symbiont cells with Fiji (ImageJ).

16. Centrifuge for 5 minutes at 5000rpm at 4°C.

17. Remove the supernatant thoroughly.

18. Add 1ml 95% Acetone.

19. Vortex.

20. Incubate at 4-8°C (in the dark) for 8 - 24 hours.

21. Centrifuge for 5 minutes at 5000rpm at 4°C.

22. Check Optical Density (at wave lengths 630, 647, 664, and 697 nm) in a 96-well plate using 95% Acetone as blank (triplicates of 200 ul per each sample). I calculated chlorophyll a concentrations according to the equation of Ritchie (2008): Chl–a= –0.3319(A630) – 1.7485(A647) + 11.9442(A664) – 1.4306(A691)