Coral micropropagation is still in its infancy. Optimizing protocols for polyp bail-out is key to fill this gap allowing scientists to use polyps as models to study corals in laboratory environments. Polyp bail-out makes it possible to create multiple propagates from small coral fragments.
Polyps can be used in different experiments facilitating the observation of microscopic processes in corals, for instance. Coral reefs are threatened. Our goal is to use this replicable model to develop strategies to protect corals from bleaching and other environmental threats in an efficient and noninvasive way.
This method could assist the investigation of coral physiology, host microbiome interactions and the mechanisms involved in bleaching. Such findings can contribute to the optimization of coral probiotics, for example. Using seawater with an adequate salinity is key.
Salinity must start at the level of the coral's natural environment and increase slowly so the stress is not excessively harmful. Collecting polyps at the correct moment and choosing those that are the most intact is crucial for their survival. A visual demonstration can be vital to understand these cues.
First, use diagonal cutting pliers to cut small fragments from a coral colony, then place them into small Petri dishes filled with 12 milliliters of seawater to which the coral colony has been acclimatized. Leave the plate open for about 24 hours at ambient temperature so that the water evaporates and its salinity gradually increases. When the tissue digestion is observable between the polyps, use a 1-milliliter transfer pipette to create a gentle flow close to the coral tissue.
The flow will slowly help complete the detachment of polyps that have already digested the tissue around them from the skeleton, then with the pipette, slowly exchange the water in the Petri dish with isosmotic water. To prepare high-salinity seawater, take normal seawater and add sodium chloride to seawater to prepare three liters of high-salinity seawater until the salinity increases by 85%After cutting small fragments of coral as demonstrated previously, put them in a 10-liter container with three liters of isosmotic seawater connected to a peristaltic pump. Fill this container with three liters of previously prepared high-salinity seawater for 24 hours at a rate of 126 milliliters per hour to increase the salinity by about 40%Using a pipette, create a gentle flow of water to release the polyps from the skeleton.
Slowly exchange the water in the container with isosmotic seawater. Add 26.29 grams of sodium chloride, 0.872 grams of potassium chloride, 2.16 grams of magnesium sulfate, 11.94 grams of magnesium chloride, 3.42 grams of sodium sulfate, and 0.26 grams of sodium bicarbonate to one liter of deionized water to prepare calcium-free seawater, then fill Petri dishes with this calcium-free seawater. After cutting small coral fragments, submerge them in calcium-free seawater in the Petri dishes.
Put the dishes in an orbital incubator for three hours at a rotation speed of 80 rpm, then transfer the fragments to Petri dishes filled with 20%DMEM prepared with artificial seawater of 40 practical salinity units and containing 100 milligrams per liter of ampicillin. Incubate the fragments at 26 degrees Celsius and 80 rpm. Exchange the media every day until tissue digestion is observable between polyps and individual polyps start detaching from the skeleton, then transfer the polyps to sterilized seawater and incubate for one hour.
Once the polyps are returned to filtered seawater with non-stressing salinity, select the viable polyps by observing tissue integrity and movement caused by ciliary flow under a stereo microscope. Place the selected polyps in a Petri dish and cover the Petri dish with a plankton net of 200-micrometer mesh size so that the polyps do not float away from the dish. Place the Petri dish inside an aquarium with appropriate conditions for the coral species used.
To prevent algae overgrowth, open the Petri dish at least once a week to renew the water and clean the dish. After selecting the polyps as shown earlier, using a transfer pipette, place them into a 75-square centimeter surface cell flask filled with 50 milliliters of isosmotic seawater, then close the flask and put them into an incubator set at 12 hour light cycles, 26 degrees Celsius and 40 rpm. If the flask gets filled with algae or biofilms, transfer the contents to a clean flask.
In this study, polyp bail-out was induced by three different methods. The water evaporation method resulted in a complete bail-out within 24 hours of incubation, and the salinity increased from 40 to 59 practical salinity units after 24 hours. The salt water supply method also resulted in polyp bail-out after 24 hours of incubation.
Here, the salinity increased from 40 to 52 practical salinity units after 12 hours of incubation, and then to 59 practical salinity units after 24 hours. The bail-out induction through incubation in calcium-free seawater was complete after three hours of incubation of polyps in it, followed by 20 hours of incubation in the 20%DMEM media, In all three methods, individualized coral polyps could be generated. Coral polyps generated by the evaporation method could survive for six weeks, whereas those produced by the high-salinity seawater supply method could survive for eight weeks in Petri dishes inside aquariums.
Polyps obtained from the seawater evaporation method kept in cell culture flasks inside incubators survived for up to three weeks without the complete dissociation of their tissues. When detaching polyps from the skeleton, it is important to be gentle. If they are not completely detached, forcing detachment by strongly pipetting water will cause damage and lower survival rates.
Using these methods, the settlement of polyps can be induced. Polyps settlement can answer questions about its calcification and growth processes. After the development of polyp bail-out induction, researchers were able to study the initial formation of the coral skeleton and directly visualize coral bleaching in the polyps scale.
