Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.
Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.
Scleractinian or reef-building corals are cnidarians capable of forming carbonate skeletons, creating reefs, and structurally complex ecosystems that can be found from deep to shallow water environments1. Tropical coral reefs host high biodiversity and provide essential ecosystem services, such as coastal protection and fisheries maintenance2. Most shallow-water reef-building corals rely on a mutualistic relationship with algae of the family Symbiodiniaceae, which provide the energy that corals require to build their skeletons. The symbiosis between the coral and the algae can be broken by environmental stress, causing coral bleaching3,4,5,6. Recent temperature anomalies have caused major coral bleaching events around the world, leading to mass coral mortality and permanent reef degradation7,8,9,10,11. As this phenomenon is based on the expulsion of symbionts by post heat stress-associated cellular mechanisms, such as apoptosis, autophagy, and exocytosis, coral bleaching can be described as a cellular process that has ecosystem-scale consequences5,6,12, which means having in vitro cultures of coral cells or tissues would be applicable to study this phenomenon closely.
Due to the importance of coral reefs and the major threats they have been facing, particularly in the past two decades2, corals have become the focus of research for protection and restoration purposes worldwide13. However, the development of approaches and experimental systems that are reliable, reproducible, and offer minimal environmental impact to study corals is a major struggle in this field.
Micropropagation is defined as the in vitro proliferation of an organism's genotype by culturing its biological material in controlled vessels14,15. The culturing of cells, tissues, and organs has been crucial for plant and animal biology over recent decades. It allows the mass reproduction of organisms in laboratories, the rapid assessment of different treatments (such as drugs and pharmaceuticals), and the direct study of cell function14,15,16,17. In general, in vitro models have been useful for complementing and deepening the studies of different organisms under better-controlled physical and chemical conditions. Due to the advantages of in vitro culturing techniques, different animal cell and tissue culture technologies have been developed, optimized, and used as important tools in many research fields, where multiple cell lines have been studied and commercialized for numerous applications16,17,18.
Many advancements in the knowledge of cell and tissue culture have been made since the first animal tissue culture in 188217, such as the use of natural and synthetic media, the invention of established cell lines, and the development of 3D media to cultivate a multitude of cell types in a better way16,17,18,19. However, the field of cell biology has mostly focused on a select group of model organisms, while many taxa still do not have well-established in vitro cultures of cells, tissues, or organs20. For instance, in coral research, no immortalized cell lines have been extensively used for research, constraining coral cell research to the use of primary cell cultures. These cultures have viability limited to a few weeks21, with no studies recording the survival of individual cells from all coral tissues for more than 13 days until the beginning of 202122. The first report of sustainable coral cell lines to be published was with Acropora tenuis cells that lived up to 6 months, and the utility of these cells for future research remains to be explored23.
To overcome the limitations in culturing coral cell cultures and to maintain a laboratory culture that preserves the overall tissue organization of corals, the use of isolated polyps has recently been proposed as a model for coral biology research24,25. Polyps are the anatomical units of corals, and each of them has a mouth located in the center of their oral disk and is connected to other polyps by the coenosarc in its aboral region26. The separation of live polyps occurs naturally by the process of polyp bail-out, in which acute stress causes the digestion of the coenosarc between the polyps, which can then detach from the colony's skeleton25,27,28. This phenomenon has been reported to occur in a variety of taxa, including octocorals29,30,31, black corals32, and scleractinian corals25,27,28,32,33, and has been linked to multiple environmental stressors, such as lack of calcium in water24,34, increased acidity35, hyperosmotic conditions25,27,32,36, high temperatures36,37, starvation33, air exposure25,30, and insecticide contamination28,38. Polyp bail-out has been, for example, reported in pocilloporid corals19, which are widely distributed across the world and are commonly used as models in coral research. Species belonging to this group, such as Pocillopora damicornis and Styllophora pistillata, have generated approximately 30-40 micropropagates from a 5 mm fragment25. This number emphasizes the advantage of using polyp bail-out as a method for coral micropropagation, as it creates the possibility of generating many genetically identical individuals from a small piece of coral. The use of isolated polyps for research also has the same advantages as cell cultures regarding the possibility of being cultured in controlled lab environments, such as flasks and Petri dishes. Additionally, microfluidic platforms to maintain live polyps have demonstrated that these micropropagates can be kept in relatively cheap and easy-to-reproduce environments, with controlled water flow, surface, and temperature24,25. These microfluidics platforms can also be used to visualize live coral structures under a microscope directly24,25.
In the present article, we summarize and demonstrate the techniques that have been developed to isolate individual coral polyps from their colonies, showing how to maintain them in laboratory conditions for long-term culture. The methods discussed include polyp bail-out through hyperosmotic conditions by evaporation and pumping high-salinity seawater and incubation in calcium-free seawater.
For the present study, a colony belonging to the Pocillopora verrucosa coral species was collected from the Al Fahal reef (22.305118 N; 38.964568 E) by SCUBA by diving using a hammer and chisel. The genus of the colony was identified morphologically, and its species was classified as P. verrucosa based on previously published work, including Pocillopora from the Red Sea, indicating that, from a genetic point of view, the species present in this area is P. verrucosa39,40. Al Fahal reef is not part of a protected environmental area, and no special permits were needed for coral collection. The colony was kept in a 300 L aquarium for a month before being fragmented and having its polyps "bailed-out". The aquarium was kept at 26 °C with two aquarium heaters, three pumps, and two light sources (see Table of Materials), maintaining a 12 h light cycle. The temperature of the aquarium was maintained by connecting each one of the two heaters to a temperature controller. Light emission was programmed to start at 6 AM and finish at 6 PM, producing an irradiance curve that peaked at 12 PM with 230 µmol photons m−2s−1.
1. Polyp bail-out by high salinity after water evaporation
NOTE: This method was adapted from Shapiro et al.25. If using different species than Pocillopora verrucosa, the size of the polyps should be taken into account before determining the size of the fragment to be cut.
2. Polyp bail-out by high salinity seawater supply
NOTE: This method was adapted from Chuang et al.27.
3. Polyp bail-out by calcium-free seawater incubation
NOTE: This method was adapted from Pang et al.24.
4. Polyp maintenance in Petri dishes
5. Polyp maintenance in incubators
Polyp bail-out was induced in coral fragments belonging to a single colony of the species P. verrucosa following three different methods (Figure 1). Bail-out induced by high salinity after water evaporation was complete after 24 h of incubation of coral fragments in Petri dishes at ambient temperature filled with water initially at 40 PSU, which then reached a final salinity of 59 PSU once the process was over (Figure 2A–C,I). Bail-out by saltwater supply was also reached after 24 h of incubation in water initially at 40 PSU that reached a salinity of 52 PSU after 12 h and 59 PSU at the end of the process, after 24 h (Figure 2D–F,I). In both experiments, an increase in salinity was responsible for the induction of tissue digestion by the polyps.After 12 h, the high salinity condition caused the contraction of the polyps in conjunction with the gradual thinning of the coenosarc, ultimately causing the final detachment of the polyps after 24 h. The bail-out induction through incubation in calcium-free seawater was complete after a 3h incubation in CaFSW followed by a 20h incubation in the 20% DMEM media (Figure 2G–H). The tissue was detached from the skeleton in all three methods after pushing it away with a pipette until individualized coral polyps (Figure 3A–C) and "tissue balls" were generated.
After the detachment, the polyps from all three methods were collected and allowed to recover in seawater before being allocated to Petri dishes covered with nets or cell flasks. The polyps obtained from the evaporation and the water supply methods were maintained in Petri dishes inside aquariums and survived for 6 weeks and 8 weeks, respectively (Figure 3D and Figure 3F). These micropropagates retained the usual anatomy of polyps, presenting tentacles, basal disks, and mouths1. The polyps obtained through the incubation in calcium-free seawater had a short life span, surviving up to 1 day, after which their tissue dissociated. Polyps obtained from the seawater evaporation method kept in cell culture flasks inside incubators survived for up to 3 weeks without dissociation of tissues (Figure 3F). In all cases, even though the polyps could not attach to the substrate, they were visually healthy and maintained their color, with zooxanthellae cells still being visible inside their tissues1.
Figure 1: Schematic representation of the three different tested methodologies for polyp bail-out induction (left) followed by the illustration of two methods to maintain the acquired polyps in lab conditions (right). (A) Representation of the methodology for polyp bail-out by water evaporation. (B) Representation of the methodology for polyp bail-out by high salinity seawater supply. (C) Representation of the methodology for polyp bail-out by calcium-free seawater incubation. Please click here to view a larger version of this figure.
Figure 2: Images of the polyp bail-out process induced by three different methodologies using fragments of the coral species P. verrucosa. (A–C) A coral fragment at 0 h, 12 h, and 24 h after incubation, respectively, in a Petri dish using the water evaporation method. (D–F) A coral fragment at 0 h, 12 h, and 24 h after incubation, respectively, using the high salinity seawater supply method. (G,H) Coral fragments exposed to the calcium-free seawater incubation method before and after, respectively. The incubations in calcium-free artificial seawater were for 3 h and in 20% DMEM for 21 h. (I) Graphical representation of the salinity values in PSU of the seawater over time during the water evaporation and high-salinity seawater supply bail-out induction methods. Please click here to view a larger version of this figure.
Figure 3: Images of P. verrucosa polyps obtained from the three demonstrated bail-out induction procedures. (A–C) The images of the polyps obtained from the evaporation, saltwater supply, and calcium-free seawater methods, respectively, were captured immediately after they were detached from the skeleton. (D) The image of a coral polyp obtained from the evaporation method after surviving 6 weeks in a Petri dish. (E) The image of a coral polyp obtained from the saltwater supply method after surviving 8 weeks in a Petri dish. (F) The image of a coral polyp obtained from the evaporation method after surviving 3 weeks in a cell culture flask. Please click here to view a larger version of this figure.
The polyps survival rate after being submitted to bail-out processes and the time needed for the process to be completed vary among previously reported research25,33,41, which is possibly explained by the different experimental approaches applied in each study. For instance, different coral species, or even corals from the same species but acclimated to different environmental conditions (e.g., corals from the Red Sea), present different thresholds to salinity levels. The method of bail-out selected and the laboratory/aquarium conditions also play important roles in the results. In some cases, the maintenance of coral micropropagates under laboratory conditions has surpassed the survival time of coral cell cultures by reaching months of survival in azooxanthellate33,41 and zooxanthellate25 corals. The time for the polyp bail-out process to be complete has also varied in different studies, ranging from a few hours25,27,30 to weeks35 of incubation exposed to the stressor responsible for causing bail-out. Another variable to be taken into account when studying polyp bail-out is the recovery of the polyps after exposure to the acute stress that triggered their release. It is still debatable if polyps after bail-out are in a good enough condition to be used as models to study coral biology. The recovery of their tissues after the degradation of the coenosarc is a matter of concern when using these micropropagates. However, polyps in many studies, including the present, have been able to present zooxanthellae cells inside their tissues and external morphologies with intact oral-aboral polarization and tentacles weeks after bail-out25,27,32,36. Previous studies have also found that, after being relieved from acute stress, released coral polyps exposed to highly saline or heated seawater have been able to recover the expression of genes related to processes such as apoptosis, proteolysis, and cell division to levels similar to the ones found before bail-out32,36 and even to increase the expression of genes related to tissue healing36.
Concerning the difference in survival between methods, it is important to highlight that this time can vary among different experiments even if the same techniques are used, and it can be related to the health of the fragments used and the proper maintenance of the polyps after the bail-out process. In the case of the bail-out through calcium-free seawater incubation, the polyp survival was limited to 1 day. Thus, it can be concluded that the method is not well suited for the long-term survival of the species studied, or a better adaptation of the technique for P. verrucosa corals from the Red Sea must be made. The reported results showed a longer survival time was obtained with the methods based on the gradual increase in salinity, when the polyps were subjected to the dripping of high-salinity water. This method can deliver a more controlled increase in salinity than the evaporation method, at the same time as not being responsible for an increase in the concentration of other substances found in the seawater, including the coral's metabolic waste, which is potentially toxic for the organism. For all these reasons, this method has been suggested as a safer alternative for maintaining healthy polyps27. Although this method has been hypothesized to be safer for polyp health and capable of producing polyps that live longer, a fact that was corroborated in this current publication, additional surveys are needed to confirm it. Both high salinity-induced bail-out experiments demonstrated a complete detachment of polyps after the salinity reached 59 PSU in 24 h. If salinity is increased past the level at which the bail-out is complete, further stress will be caused to the polyps, reducing their chance of surviving and recovering from the acute stress treatment. Therefore, it is not recommended to maintain the polyps longer in such salinity levels. When performing the bail-out induction method by exposure to calcium-free seawater, a complete detachment was obtained from a 3 h incubation in calcium-free artificial seawater, meaning further exposure to this medium is also not recommended.
To address the methods that were more adequate for the study of coral polyps in laboratory/in vitro surveys, this study focused only on three procedures that took close to 24 h for the bail-out process to be complete and were used in studies that involved the long-term maintenance of coral polyps from scleractinian corals. Other methods reported to take significantly longer than this time were not engaged. The settlement of polyps to a substrate was not attempted in this study, which focused on producing polyps that could be transferred to different environments or easily collected for analyses using disposable pipettes. The results demonstrate that polyps from the coral species P. verrucosa were kept alive, with associated zooxanthellae cells, healthy visual status, and a preserved gross external anatomical structure, for up to 8 weeks, even without attachment to a substrate. These results indicate that more biological replicates can be generated from single coral fragments using some of the techniques demonstrated in this study. Such biological replicates can be kept in controlled environments (such as Petri dishes and cell flasks) and maintained in laboratory conditions for month-long experiments and used for several purposes.
Since the first incidental descriptions of polyp bail-out42,43, new protocols have been established to find more standardized methods for inducing polyp release and maintaining such polyps alive, which can be used for future research applications. These include investigating different aspects associated with the coral holobiont physiology44 and host-microbiome interactions45, the molecular mechanisms involved in coral bleaching5,25, and the health, resilience, and protection of the coral holobiont12,13,46,47. Moreover, released coral polyps can be used for applications outside the realm of research and have been suggested to be useful for creating propagules that can attach to a substrate and grow, potentially creating multiple coral individuals that can be used for restoration purposes once standardized protocols for bail-out become widespread28. Overall, although more in-depth experiments using bailed-out polyps should be performed to standardize the methodology, it has been shown that polyp bail-out is a reproducible approach that can be applied as a tool in coral research for several purposes.
The authors have nothing to disclose.
We thank Adam Barno and Francisca Garcia for their support in the experiments and monitoring of the coral polyps. We also thank the KAUST Coastal & Marine Resources Core Lab for their assistance regarding the aquarium maintenance and infrastructure. The study was funded by KAUST grant number BAS/1/1095-01-01.
5560 Conductivity/Temperature Probe | YSI | 5560 | Conductivity probe used with the ProQuatro Multiparameter meter |
Ace 5 in. Alloy Steel Diagonal Pliers | Ace Hardware | 2004083 | Used to cut coral fragments |
Ampicillin sodium salt | Sigma-Aldrich | A9518 | Used in DMEM medium. |
DMEM (1x) Dulbecco's Modified Eagle Medium | Gibco | 41965-039 | Used for incubating coral fragments in the calcium-free polyp bail-out method |
Fisherbrand Petri Dish, Stackable Lid 60 mm x 15 mm Sterile, Polystyrene | Thermo Fisher Scientific | FB0875713A | Petri dish used for bail-out by evaporstion and for keeping polyps inside an aquarium. |
Heizer Titanrohr Heizstab SW MW 600 Watt | Schego | 548 | Heaters used in aquarium |
Leica Application Suite Version 4.2 | Leica Microsystems | NA | Software used for image capture in demonstrative results |
Leica IC80 HD | Leica Microsystems | 12730216 | Camera used to take demonstrative results pictures |
Leica MDG33 | Leica Microsystems | 10 450 123 | Stereoscope stand used to take demonstrative results pictures |
Leica Z6 APO | Leica Microsystems | NA | Macroscope used to take demonstrative results pictures |
Magnesium Chloride | Thermo Fisher Scientific | 7487-88-9 | Used for preparing calcium-free artificial seawater. |
Magnesium Sulfate Anhydrous | Sigma-Aldrich | 7791-18-6 | Used for preparing calcium-free artificial seawater. |
Masterflex I/P Easy-Load Pump Head for Precision Tubing, White PPS Housing, SS Rotor | Masterflex | HV-77602-10 | Peristaltic pump head. |
Masterflex L/S Precision Modular Drives with Benchtop Controller | Masterflex | EW-07557-00 | Peristaltic pump drive used for pumping high salinity seawater. Can be substituted for any peristaltic pump capable of mainaining water flow as described in protocol. |
Masterflex L/S Precision Pump Tubing, Platinum-Cured Silicone, L/S 16; 25 ft | Masterflex | HV-96410-16 | Tubing for peristaltic pump. |
Millex 33 mm PVDF 0.22 µm Sterile RUO | Sigma-Aldrich | SLGVR33RS | Used to filter artificial sea water. |
Nunc EasYFlask 75 cm2 Nunclon Delta Surface | Thermo Fisher Scientific | 156499 | Flask usually used for cell culture used for polyp culture. |
Orbital shaker, Advanced 5000, VWR | VWR | 444-2916 | Shaker used inside incubator. |
Percival Incubator – I-22VL | Percival | NA | Incubator used for maintaing corals kept in cell flasks. |
Plankton net 200 µm mesh size | KC Denmark | NA | Used for covering petri dishes containing coral polyps. |
Potassium Chloride | VWR Chemicals | 7447-40-7 | Used for preparing calcium-free artificial seawater. |
ProQuatro Multiparameter Meter | YSI | 606950 | Used for measuring salinity thoughout the protocol |
RADION XR15 G5 PRO | Ecotech | NA | Lights used in aquarium |
Red Sea Salt Premium grade, moderate Alkalinity |
Red Sea | NA | Used to prepare 40 PSU artifical sea water. |
Sodium Bicarbonate | Sigma-Aldrich | 144-55-8 | Used for preparing calcium-free artificial seawater. |
Sodium Chloride | Sigma-Aldrich | S3014 | Used for preparing calcium-free artificial seawater. |
Sodium Sulfate Anhydrous | VWR Chemicals | 7757-82-6 | Used for preparing calcium-free artificial seawater. |
TRD 112 thermostat | Schego | NA | Thermostat used in aquarium |
Turbelle Nanostream 6025 | Tunze | 6025 000 | Pumps used in aquarium |