Jan 16, 2025
Cloning method for Symbiodiniaceae from Corals
This protocol is a draft, published without a DOI.
- Nathan Chen1,
- Tze Ching Yong1,
- Jih-Terng Wang1
- 1National Sun Yat-Sen University, Taiwan
Protocol Citation: Nathan Chen, Tze Ching Yong, Jih-Terng Wang 2025. Cloning method for Symbiodiniaceae from Corals. protocols.io https://protocols.io/view/cloning-method-for-symbiodiniaceae-from-corals-dxzn7p5e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 16, 2025
Last Modified: January 16, 2025
Protocol Integer ID: 118542
Keywords: cloning coral symbiotic dinoflagellate, coral symbiotic dinoflagellate, cloning method for symbiodiniaceae, several species of coral symbiont, coral symbiont, symbiodiniaceae, coral, bleaching stress, cloning method
Funders Acknowledgements:
National Council of Science and Technology, Taiwan
Abstract
A procedure for cloning coral symbiotic dinoflagellates was developed in this study. Several species of coral symbionts were
successfully cloned, with two of them further characterized. Experiments with the two species isolated from Turbinaria sp.
showed that damage from light intensity at 340 μmol photons/m²/s was more severe than from high temperature at 36 °C. Additionally, preincubation in high salinity conditions activated their endogenous tolerance to bleaching stress. Pretreatment at 50 ppt salinity reduced the percentage of cells stained for ROS by 59% and 64% in the two species under bleaching stress compared to those incubated at 30 ppt. Furthermore, their Fv’/Fm’ during the recovery period showed a significant improvement compared to the controls. (A part of the abstract from the reference.)
Troubleshooting
Cloning of Symbiodiniaceae from Corals
For more details of this protocol, please reference "Activation of endogenous tolerance to bleaching stress by high salinity in cloned endosymbiotic dinoflagellates from corals" authored by Chen, Yong and Wang in Botanical Studies 2025.
Rinse a piece of healthy coral using artificial seawater. Smash coral polys by spreading high pressure artificial seawater using a painter's brush hooked to an air compressor.
Dilute the mix of coral debris and symbiotic dinoflagellates (Symbiodiniaceae) to 25 mL, filter the suspension using a layer of Kimwipe paper supported by a layer of 25 micro-meter mesh nylon filter.
Settle the dinoflagellate cells using low speed centrifugation (100 X g for 10 min) in a 50 mL tube. Remove the supernatant by suction. Repeat this cleaning step once.
Re-suspend the dinoflagellate cells in 40 ppt artificial seawater containing 36 mg IMK nutrient/L and 200 micro-molar glycine. Adjust the cell density to no more than 1 cell per 200 micro-liter.
Transfer 200 micro-liter of the cell suspension to each well of medical grade 96-well plates. One hundred micro-liter of the same medium was added to each well to make 300 micro-liter per well.
Keep these plates at 25 degree C in the dark for the first week, then move them to 20 micro-mole photon/m2/sec (day/night = 14/10 hr) at 25 degree C. Evaporated water in the wells is refilled twice per week. The incubation takes about 3 months before significant growth can be observed under an inverted microscope.
When significant amount of dinoflagellates are observed in some wells, change the medium to 40 ppt artificial seawater with 250 mg IMK nutrient/L. Increase the light intensity to 50 micro-mole photon/m2/sec.
When cell clumps are visible by naked eyes, transfer these clones to 24-well medical grade plates and bring the volume to 2 mL to continue their growth.