TITLE: Novel Ex-situ Stony Coral Tissue Loss Disease Treatment Protocol
AUTHORS: Gregory Pelose, Macarena Blanco-Pimentel, Johanna Calle-Triviño, Ariadna Leon, Victor Galván, Stephanie Schopmeyer, Delaney Foster, Erika Harms, Cullin Burdett, Megan K. Morikawa

[DESCRIPTION]
Stony Coral Tissue Loss Disease (SCTLD) is a highly lethal coral tissue loss disease that has shown to cause catastrophic loss of coral cover and biodiversity in Caribbean coral reef ecosystems. The unprecedented loss to SCTLD has demonstrated significant harm to reef functionality, making intervention and mitigation strategies essential. Current in-situ and ex-situ treatment methods have shown varying levels of success. Here, we present a 10-day ex-situ treatment protocol demonstrating a 94% success at treating coral colonies across four genera with SCTLD-like lesions (n=34 with Dendrogyra cylindrus, Montastraea cavernosa, Pseudodiploria strigosa, Orbicella faveolata, O. annularis, and O. franksi) with a 0% reinfection rate after treatment (ex-situ) (Pelose et al., In prep). The three main components of the treatment include a pre-cleaning procedure to remove necrotic tissue, hydrogen peroxide (H2O2) dips to oxidize potential pathogens on exposed skeleton and the coral's outer tissue layer, and a multicomponent antibiotic treatment dip to eliminate any remaining potential pathogens. This protocol explains how to conduct this novel treatment and provides recommendations for coral collection and preparation methods, coral husbandry, and post-treatment care.

[STEPS]
SECTION: Reasoning for Protocol
1. Stony Coral Tissue Loss Disease (SCTLD) was first observed in 2014 near Miami, Florida and has rapidly spread across the Caribbean (Precht et al. 2016; Walton et al., 2018; Estrada-Saldívar et al., 2020). SCTLD affects at least 22 species of Caribbean stony corals and has resulted in the loss of up to 60% of coral cover in many Caribbean regions following initial exposure (Brandt et al., 2021; Heres et al., 2021; Papke et al., 2024). The opportunistic behavior of SCTLD is exacerbated when in contact with highly susceptible species, with as high as 90-100% reduction in local abundance reported within months of exposure (Walton et al., 2018; Estrada-Saldívar et al., 2020; Papke et al., 2024). This unprecedented loss of coral cover has been shown to reshape reef functionality (Alvarez-Filip et al., 2022) and has resulted in an urgent need for effective intervention and mitigation strategies to combat SCTLD and preserve genetic diversity of susceptible coral species. Current in-situ and ex-situ treatments have shown varying levels of success at halting the progression of SCTLD-like lesions in coral colonies (Miller et al., 2020; Walker et al., 2021; Forrester et al., 2022; Studivan et al., 2023; Papke et al., 2024). There are currently no published protocols available to effectively treat affected coral tissue in ex-situ facilities. The authors here seek to address this gap by presenting a 10-day treatment method that has shown to treat Caribbean coral colonies displaying signs of SCTLD with a 94% success rate (Pelose et al., In prep). The methods for this protocol were derived from treatments and resources within the reef-keeping hobbyist community and have been tested with 34 coral colonies from 6 Caribbean coral species (Dendrogyra cylindrus (n= 14), Montastraea cavernosa (n= 8), Pseudodiploria strigosa (n= 4), Orbicella faveolata (n= 3), Orbicella annularis (n= 2), and Orbicella franksi) (n= 3)).

SECTION: Coral Collection and Preparation
2. Coral collection, transport, and labeling:

This protocol suggests that when collecting Caribbean coral showing similar conditions to those of SCTLD, tissue near active lesions should be prioritized for collection as opposed to areas of 100% apparently healthy tissue (see Figure 1 for examples). Prioritizing active lesions and their surrounding live tissue, and thus removing active disease from the environment, may reduce potential pathogen load and support the ecosystem (Prentice et al., 2019; Papke et al., 2024). Once donor colonies are located, fragments showing signs of SCTLD can be removed and immediately placed and sealed in ziplock bags. When possible, epoxy is applied to open lesions left on the donor colonies after collection. Bags should remain sealed to minimize potential contamination during transport to a boat or shore-based location where they can then be placed in a transport vessel filled with seawater. Once the sealed bags have been placed in their transport vessel and contamination risk is no longer present, the bags should be opened during transport. Fragments should then be brought to a temporary quarantine holding system within a land-based holding facility in a timely manner.

 

Prior to starting the treatment protocol, also referred to as the Treatment, colonies should be trimmed of excess exposed skeleton, wherever possible, and attached to a labeled base for easier transport and identification throughout the Treatment (Figure 2A). The labeled base should allow for the colony to be picked up and moved into containers without physically touching living tissue (Figure 2B). While attaching the corals to their respective base, using epoxy or super glue, it is important to minimize covering up exposed skeleton. Covering the skeleton may create a reservoir of pathogens or nuisance organisms that will not be mitigated by the Treatment. These reservoirs may allow continued exposure to potential pathogens or nuisance organisms during treatment or post-treatment and negatively impact coral health. Once the colonies are attached to their bases, they can remain in their quarantine tank until commencing the Treatment. To prevent further advancement of tissue loss, the Treatment should begin the day after collection, but can begin later if necessary. Trials have shown the Treatment to still be successful up to 26 days after collection (Pelose et al. In Prep).

 

SECTION: Coral Collection and Preparation
3. Quarantine and post-treatment holding system set-up:

Corals need to be held in a holding system that has shown the capability to support long term coral growth and recovery prior to the potential stress of the Treatment. If the holding system has not shown the capability to successfully hold and grow corals, it may result in a decreased functionality of the Treatment. Water quality parameters should stay stable and ideally be maintained within the “Ideal Parameters for Reef Aquariums” reference chart (https://www.bulkreefsupply.com/content/post/md-2021-03-reef-tank-parameters). It is best to replicate PAR values recorded at the collection site or this protocol recommends providing your corals with an average of 200 PAR during the 10 day treatment. Antibiotic treatments should be started at a time that minimally affects the normal photic period of the colonies being treated (approximately 2 P.M. or later is recommended).

Corals should be held within a temporary quarantine holding system until the treatment starts. After day one of the Treatment, colonies will be moved into a post-treatment holding system where they will reside for the remaining 9 days of treatment and thereafter. There are two options for a post-treatment holding system (Figure 3). The first, and preferred option, includes moving the colonies into a separate, clean system that does not have other coral colonies in it. If a separate system is not available, it is still possible to conduct the treatment by modifying the original quarantine tank (Figure 3, Option Two) during day one of treatment. Modifications include using a siphon hose to clean detritus from the tank until 50% or more of the water is removed. This modification needs to occur during the first four-hour antibiotic treatment dip conducted on Day 1. Experimental trials for this protocol were conducted in a flow-through, open system utilizing Option Two (Figure 3), where the original quarantine holding system was modified during day one of treatment and then utilized as the post-treatment holding system.

 

SECTION: Coral Feeding
4. While in holding and during the treatment schedule, colonies should receive a consistent feeding schedule to support tissue growth and overall coral health. This protocol suggests feeding once a day with one and a half teaspoons (tsp) of Reef-Roids (https://www.polyplab.com/) coral food mixed in approximately 300 mL of saltwater coming directly from the holding system the corals are held in (Figure 4).

 

The total amount of this mixture is dependent on the quantity of corals being fed and should be calculated accordingly. For reference, 300 mL of mixture can feed approximately 60 colonies of similar size to the Montastraea cavernosa colony in Figure 11 at day 349 (approximately 33 cm2). The Reef-Roid solution should be mixed thoroughly with a baster and allowed to hydrate for a minimum of 15 minutes prior to feeding. While waiting for the coral food mixture to hydrate, all internal flows of the holding tank should be turned off including any form of internal pump, wavemaker, and flow that may inhibit coral feeding. At the end of the hydration process, thoroughly mix the food again with the baster and target feed each coral (see Video 1 for reference). All corals in a tank should be fed this mixture and carefully monitored to ensure the majority of corals are eating (see Video 2 for reference). Once all corals have been target-fed, they should be allowed to digest for 45 minutes to 60 minutes. If any coral is not observed to feed during the initial feeding, those individuals should be target-fed again after all corals are fed. All colonies should be periodically monitored throughout the digestion period. The feeding preparation should be started at least 1.5 hours prior to treatment and all water flow should remain off during this period. Since treatment is suggested to start at or after 2:00 P.M. to minimize changes to the coral's normal photic period, feeding should start at 12:00 to 12:30 P.M. or later.

This concentration of coral food has successfully supported corals undergoing the Treatment, but at this time, has only been conducted in an open system with a constant influx of new seawater. The constant incoming flux of seawater likely aids in the system’s ability to manage this concentration of nutrient load. As such, conducting this feeding regime within a closed system may result in a significant change to water quality and subsequent water quality monitoring, water changes, and tank maintenance should be increased to reduce the impacts of high nutrient loads.

 

Video 1: Example of target feeding a Dendrogyra cylindrus colony 108 days post-treatment.


 

Video 2: Timelapse video of a Montastraea covernosa colony actively eating 145 days post-treatment. This 23 second video condenses 18 minutes of feeding activity during the digestion period. 
SECTION: Treatment Materials
5. The 10-day Treatment includes a pre-treatment hydrogen peroxide (H2O2) dip, an antibiotic/antiseptic treatment dip, and a post-treatment hydrogen peroxide dip each day. For hydrogen peroxide dips, three percent hydrogen peroxide and a 50 mL to 100 mL graduated cylinder are required (Figure 5). The antibiotic/antiseptic treatment requires three dry antibiotic reagents and two liquid reagents (Figure 6). The three dry reagents are amoxicillin, ciprofloxacin, and an aquarium treatment product called Chemiclean (https://chemi-pure.com/products/chemiclean/). Local procurement procedures should be followed to obtain antibiotics (i.e., may require a permit and/or prescription). The two liquid reagents are Lugol’s solution (iodine antiseptic) and Restor (coral tissue regrowth supplement), both made by Brightwell Aquatics (https://brightwellaquatics.com/products/index.php). The concentrations of each reagent used for a four gallon treatment can be found in Figure 6, but can be adjusted as needed for larger or smaller treatments to accommodate the quantity or size of colonies being treated (Table 1). Five containers with the same dimensions will be needed to conduct this protocol (Figure 7). It is also important to note that a four gallon treatment will utilize up to 20 gallons (75.7 L) of seawater during each day of the Treatment. If a closed system is being utilized, it is recommended that seawater is pre-mixed and prepared to refill the holding system after each day of treatment.

 

 

 

Table 1. Concentrations of reagents for one gallon, four gallon and eight gallon treatment solutions.
 

SECTION: Treatment Preparation
6. Antibiotic treatment dip and pre-treatment H2O2 dip preparation:

The antibiotic treatment dip container should be prepared first to allow time for all reagents to dissolve. Collect four gallons (15.1 L) of seawater in a treatment container (Figure 6B) from the same tank or water source where the corals are held. Do not fill the treatment container completely as it is important to allow volume to add the corals. The small internal filter (Dophin Internal Filter KF-350 with sponge) is added next to start circulating the water and remove particulates that may be floating throughout the antibiotic treatment dip. A 100-watt heater can also be added into the antibiotic treatment dip container to maintain the water temperature above 23.3 °C (74 degrees Fahrenheit).

In the following order, mix the dry materials, Ciprofoxacin (500 mg), Amoxicillin (500 mg), and Chemiclean (10 level scoops (0.15 cc) = 1.5 cc total) into the antibiotic treatment dip container. Adding the ciprofloxacin first allows the hard 500 mg tablet to dissolve over time. Next, the internal powder contents of the 500 mg amoxicillin capsule is added to the antibiotic treatment dip container being careful to not allow water to come into contact with the capsule. Add 10 level scoops of Chemiclean (use a flat utensil to level off the powder inside the scoop to reduce variability). After adding the Chemiclean, use the kitchen baster to mix the surface of the water until all floating dry reagents are inundated to ensure that liquid reagents do not bind with them on the surface. Once there are no longer dry materials visibly floating on the surface of the water, add the liquid reagents in the following order: 1) Lugol’s iodine solution (40 drops) and 2) four capfuls (7 mL each for a total of 28 mL) of Brightwell’s Restor. Once all reagents are added, the solution should be left to continue dissolving for 20-30 minutes. The solution should be mixed with the kitchen baster roughly every 5 minutes to assist the dissolving process. Note that it is not uncommon to have some small undissolved particles present in the bottom of the container at the end of the wait time.

While the antibiotic dip is dissolving, prepare the pre-treatment H2O2 dip by collecting three gallons (11.4 L) or four gallons (15.1 L) of tank water into another treatment container. The water level should be high enough that all the corals will be fully immersed when added into the container. Next, add 40 mL of 3% H2O2 per gallon (3.8 L) of seawater. This prepared pre-treatment H2O2 dip container can be placed next to the dissolving antibiotic treatment dip container.

SECTION: Start of Protocol
7. Pre-cleaning:

Fully submerge the transport container into the holding system that the corals are in and carefully place all treatment colonies into the submerged container without exposing them to air. Attempt to arrange the colonies evenly and in an order that can be duplicated within the H2O2 dip container and antibiotic treatment container. Carefully remove the transport container from the holding system with the colonies inside and set it next to the pre-treatment H2O2 dip container and antibiotic treatment dip container. If the holding system does not have ample space to submerge the transport container inside, then the transport container may be filled with tank water using a siphon hose. Colonies should then be placed into the filled transport container one by one, ensuring that each colony is first lightly shaken in the tank to promote tentacle retraction prior to being temporarily removed from the water. 

Within the transport container, all corals should receive a pre-cleaning with water pressure from a kitchen baster to remove any necrotic or dead tissue near or on active SCTLD-like lesions. It is important to only apply enough water pressure to remove the tissue that has become detached and avoid damaging the living tissue still attached to the skeleton. Depending upon the progression of the lesion, up to an inch of decaying tissue may come off from active disease lesions. The transport container may become clouded, and the decaying tissue and potential pathogens may produce a foul smell. In many cases, it will not be possible to remove all necrotic or dead tissue detached from the skeleton.

SECTION: Start of Protocol
8. Pre-treatment hydrogen peroxide (H2O2) dip:

After pre-cleaning, use a clean kitchen baster to stir the contents of the prepared pre-treatment H2O2 dip container immediately prior to adding the corals to prevent layers from forming in the container. In addition, the antibiotic treatment dip should also be stirred one last time to allow any undissolved reagents time to settle to the bottom of the container. Coral colonies should be shaken underwater while still in the transport container to remove any particulates that rest on top of the colony post pre-cleaning and also to promote the retraction of polyp tentacles before being removed from the water. Place each coral into the pre-treatment H2O2 dip container for 10 minutes and record the order that the colonies are added so they can be removed in the same order. It is important to let the corals sit for the full 10 minutes to allow for the H2O2 to oxidize and disinfect the exterior of the coral tissue, exposed skeleton and labeled ceramic base. During this process, micro-bubbles may form on the colonies and may be concentrated on the exposed skeleton created during cleaning (Figure 8A). It is common for colonies to expel waste (Figure 8A) or temporarily expose mesentery digestive filaments (Figure 8B) during the pre-treatment H2O2 dip. At the 10-minute mark, colonies should be lightly shaken under the water to remove microbubbles and exposed skeleton should be cleaned using water pressure from the kitchen baster. Colonies should then be removed from the pre-treatment H2O2 dip in the order that they were originally added.

 

SECTION: Start of Protocol
9. Antibiotic treatment dip:

Corals should be placed directly from the pre-treatment H2O2 dip container into the antibiotic treatment dip container in the same order that each colony was added to the pre-treatment H2O2 dip. Ensure that the flow coming from the internal filter in the antibiotic treatment dip container is not pointed directly at any coral tissue. Corals will soak in the antibiotic treatment dip for four hours and should be temporarily monitored, ensuring that the internal filter does not become dislodged and pointed directly at living tissue. After day one of the antibiotic treatment dip, colonies will utilize the post-treatment holding system (see Quarantine and post-treatment holding system set-up).

SECTION: Start of Protocol
10. Post-treatment hydrogen peroxide (H2O2) dip and transfer to post-treatment tank:

Prepare the post-treatment H2O2 dip near the three-and-a-half-hour mark of the antibiotic treatment dip. Fill the post-treatment H2O2 Dip container with three gallons (11.4 L) or four gallons (15.1 L) of tank water from the post-treatment holding system and add 40 mL of 3% H2O2 per gallon (3.8 L). Prior to adding the corals, the post-treatment H2O2 dip solution should be mixed with a baster to disrupt any layers that may have formed in the mixture. Post mixing, fragments can then be moved in the same order that the colonies entered the antibiotic treatment dip into the post-treatment H2O2 dip container. Colonies will remain in the post-treatment H2O2 dip for 10 minutes. If a coral has significant polyp extension while in the antibiotic treatment dip container, they should be lightly shaken so that they retract their tentacles prior to being taken out of the water and transferred into the post-treatment H2O2 dip container. While the corals are in the post-treatment H2O2 dip container, a clean transport container should be used to collect water from the post-treatment holding system where the corals will be held post-treatment. If the treatment is being conducted close to the post-treatment holding system (i.e., 5 seconds or less to transfer the corals), then a transport container is not necessary. After 10 minutes in the post-treatment H2O2 dip, colonies should be lightly shaken under the water to remove microbubbles and exposed skeleton should be cleaned using water pressure from the kitchen baster. Next, transfer the corals from the post-treatment H2O2 dip container in the order that they originally were placed into their post-treatment holding system via the transport container or directly into the tank. If a transport container is being used, it can be submerged into the post-treatment holding system if possible to avoid taking the colonies out of the water again. If it does not fit, then the colonies can be removed from the water and placed into the post-treatment holding system one by one.

SECTION: Start of Protocol
11. Cleanup and prep:

The antibiotic infused water from the antibiotic treatment container may be stored in large and labeled containers for professional disposal or it may be disposed via local regulations. The contents coming from the pre- and post-treatment H2O2 dips and the pre-cleaning may be discharged down drains that lead to sewage treatment plants or as required by local regulations. All containers should be rinsed with freshwater and set to fully dry overnight to be used again the following day. All basters and coral feeding containers can be placed in a mild bleach solution (20 mL bleach to one liter of H2O) overnight and then set to air dry the following morning to be utilized in the afternoon.

SECTION: Start of Protocol
12. Following 9 days of treatment:

The same procedure is to be repeated for a total of 10 days. All saltwater used for the rest of the treatment should come from the post-treatment holding system. Figure 9 below provides a timeline that can be followed to aid in completing each day of the Treatment.

 

SECTION: Post Treatment
13. After the final day of Treatment, the colonies can either stay in the post-treatment holding system or be moved to a different holding system. It is recommended that feeding is conducted in the same way post-treatment to help enhance regrowth over existing lesions.

SECTION: Protocol Trial Results
14. Using the protocol described above, 32 of the 34 treated colonies (94% success) survived the treatment and had no additional tissue recession post-treatment (Pelose et al. In prep). Figures 10 and 11 show examples of coral colonies that were treated with this protocol. These colonies, along with many others, demonstrated the capability to regrow over previously exposed skeleton.

 

 

A variety of colony sizes were treated using this protocol, one of the smallest being approximately 5.10 cm2 (± 0.05 cm2) of size (Figure 12). This colony had nearly an undetectable amount of tissue remaining on the skeleton after pre-cleaning (Figure 12, Day 1).

 

Therefore, the use of this protocol demonstrates that colonies containing even small portions of healthy tissue (approximately 5.10 cm2 (± 0.05 cm2)) may survive and recover. Some colonies showing signs of SCTLD were also partially bleached before going through the treatment protocol. These colonies were able to survive the treatment and also increase symbiont density post-treatment (Figure 13).

 

All treated colonies have been placed in Iberostar’s land-based restoration facility located in the Dominican Republic. These recovered colonies represent valuable genetic material as many of the mother colonies that the treated fragments came from have since fully perished. It is the hope of this publication that this treatment, if scaled, can play an important role in Caribbean coral genetic preservation while also helping other organizations keep SCTLD out of their land-based restoration and genetic banking facilities.

SECTION: Acknowledgements
15. We want to thank:

The brilliant minds of the reef keeping hobbyist community, specifically Loc Huynh (Danny) from Kung Fu Corals, for creating innovative strategies to help corals and for openly sharing them with both the hobbyist and scientific communities.
Camila Valverde from the University of Costa Rica for her enthusiasm and help with multiple post-experimental treatments.
Dressel Divers for all of the dive support, specifically Sofia Gil Estevez for her help during collection events and Paula Ventura for her incredible capability to find Pillar Coral colonies.