Materials and methods for promoting coral growth

Abstract

Competition during coral growth is mitigated by coating a structure that supports a coral fragment with a lubricant infused polymer that inhibits adhesion of unwanted organisms on the structure adjacent to the coral fragment. The structures are suitable for installation in natural (ocean) environments to support coral restoration and for use in marine reef aquaria.

Description

[0001] MATERIALS AND METHODS FOR PROMOTING CORAL GROWTH

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of the priority of U.S. Provisional Application No. 63 / 692,633, filed September 9, 2024, which is incorporated herein by reference in its entirety.

[0004] GOVERNMENT RIGHTS

[0005] This invention was made with government support under HR001121 SOO 12 awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] The present invention relates to promoting coral growth in natural and artificial environments and more particularly to a method and coating for preventing the build-up of competitive algae that can inhibit or interfere with coral growth.

[0008] BACKGROUND

[0009] Coral reefs are rapidly degrading due to rising ocean temperatures, ocean acidification, overfishing, and coastal eutrophication. Even though coral reefs go through natural cycles of disturbance and recovery, current environmental conditions have led to an increased number of disturbance events that hurdle the ability of the reef community to recover effectively. Mass bleaching events have become more frequent and prevalent in recent years. It is estimated that since the 1950s, coral reef coverage and diversity have declined by half. Coral reefs are vital components of the ecological landscape in tropical and subtropical regions and are hotspots of marine biodiversity. The global degradation of coral reefs thus has crucial implications for ocean productivity, biodiversity, and coastal resilience. Reef loss also has far-reaching economic repercussions due to their vital role in fisheries and coastal protection as well as tourism and trade. Goods and services derived from reef ecosystems amount to over $30 billion USD annually. Significant and concerted actions must be taken to reduce and prevent ongoing reef decline. Ultimately, reducing global climate threats and managing local stressors are two key strategies for managing the source of reef degradation. Local management approaches include the assignment of marine protected areas (MP As), the promotion of sustainable fishing practices, and water quality management. Unfortunately, management alone has already proven to be insufficient. The threats facing coral reefs are overwhelming and the consequences of failure will be devastating.

[0010] There is an urgent need for more active reef restoration strategies, including coral gardening and the exploration of new restoration technologies. Biologically driven approaches include increasing the thermal tolerance of corals via assisted evolution and therapeutic treatments such as probiotic and microbiome modulation. More recently, there has been a push to explore approaches from adjacent engineering disciplines, such as bioengineering and nanotechnology, in order to accelerate biodiversity restoration or to kickstart ecosystem processes for hybrid and artificial reefs. A major challenge in coral restoration and the engineering of hybrid reefs is to ensure the successful growth and propagation of outplanted corals, especially in areas where local mismanagement facilitates high algal cover and intense space competition. High nutrient levels and low herbivorous fish populations lead to elevated competition with coral fragments. Effects of such high cover go beyond immediate space competition and alter the reef biochemistry, accelerating reef hypoxia and changes in reef microbial communities. To enhance the competitive success of corals, managers have thus aimed to increase herbivorous fish populations and reduce nutrient levels to limit macroalgal productivity. Despite conservation efforts, including marine protected areas and sustainable fishing practices, the magnitude of the challenges necessitates innovative approaches for reef restoration.

[0011] Another area that could benefit from the development of improved coral growth and restoration strategies is marine aquaria: public and private installations that strive to reproduce conditions within the ocean by creating artificial reefs that support marine life - saltwater fish and invertebrates, including corals. Corals usually grow in shallow, very clear water with an abundance of sunlight and moderate turbulence. The ability to recreate a natural environment in these artificial settings requires appropriate lighting, water movement, and meticulous maintenance of water chemistry and quality. Challenges similar to those that occur in the ocean — competition for light, space, and nutrients — also exist in reef aquaria, but can be amplified in the such closed systems. Improvements that encourage the growth of corals and control competition that impacts coral growth would be a significant boon to the aquarium industry, covering the full range from small scale private aquariums to large scale public displays. SUMMARY

[0012] According to embodiments described herein, bio-inspired materials are provided to address the challenge of algal competition on coral reefs. The inventive approach employs slippery liquid-infused porous surfaces, or “SLIPS”, as a fouling-prevention coating tailored for coral reef restoration and engineering. The effectiveness of these coatings in mitigating algal competition and facilitating coral growth were evaluated through aquarium experiments and in situ trials on O’ahu, Hawai'i.

[0013] The inventive SLIPS technology is based on polydimethylsiloxane (PDMS),a nontoxic, inert, silicone-based polymer with a wide range of applications including medical devices, cosmetics, food, and microfluidics. PDMS is optically transparent, and thermally stable with hydrophobic and viscoelastic qualities. Test results demonstrate that PDMS- based SLIPS coatings significantly reduce algal coverage compared to commercial aragonite-based surfaces, with up to 70% reduction observed over a 12-week deployment period in situ. In some aspects, the inventive scheme includes the fabrication of coral-guard structures, which are slippery substrates customized for coral fragment outplanting. Coralguards facilitate tissue growth of Stylophora pistillata fragments, without competitive turf algal growth. These approaches hold promise for advancing restoration efforts, including the engineering of hybrid reefs and targeted coral gardening approaches.

[0014] The inventive scheme leverages advances in nanoengineering and materials science to develop new solutions to managing algal competition, including fouling-prevention coatings. Specifically, SLIPS provide a non-toxic technology that applies an immobilized lubricant layer that changes the foul ant- surface interactions from a solid-solid to a solidliquid interaction, therefore minimizing the adhesion of unwanted organisms in the process. While PDMS was used to test the effectiveness and efficacy of the inventive scheme, the SLIPS principle can be implemented using other materials, including, but not limited to, polytetrafluoroethylene (PTFE) and similar synthetic polymers, and steel, to create a micro- nanoporous substrate that is infused with a lubricant / oil.

[0015] A lubricant-infused 3D polydimethylsiloxane (PDMS) polymer network, i-PDMS, described by Araini et al., in Science, 357:668-673 (2017), incorporated herein by reference, was used for evaluation of the inventive approach for coral reef restoration. The suitability of this infused PDMS reef substrate, hereinafter, “reef-PDMS”, was evaluated via quantifying algal coverage in both aquarium settings and in situ in two locations surrounding the fringing reefs in O’ahu, Hawai'i. The SLIPS principle can be augmented to develop “coral -guards,” a scalable and simple solution that reduces the immediate competitive interactions between coral fragments and the adjacent benthos. Results highlight that reef- PDMS and coral-guards significantly prevent the build-up of competitive algae and can thus be applied as effective means to reduce competitive pressure on outplanted corals in coral nurseries, aquaria, and other restoration efforts.

[0016] In some aspects, the inventive PDMS surfaces ensure that the substrate will not be covered by competing algae. While coral cannot grow directly on this substrate, the reduction of overall algal growth on the artificial reef has many secondary benefits. For example, a reduction in algal growth on artificial reef implies less production of harmful toxins / metabolites by macroalgae / bacterial biofilms, and less potential bacteria in the water associated with harmful algae. Essentially, the reef-PDMS coating acts as a placeholder and ensures that algal biomass does not dominate the artificial reef. By including the reef-PDMS on specific areas in the form of plugs or tiles of a certain size, e.g. 5 cm2, if corals are in proximity they can overgrow the reef-PDMS.

[0017] The inventive SLIPS (slippery liquid infused porous surfaces) principle provides for coral restoration applications, creating a stable bonding of the PDMS with the aragonite coral plug. A placeholder in the center of the plug allows for the attachment of coral fragments. Experiments demonstrate that while the PDMS-infused surface reduces algal and competitor coverage, it facilitates the attachment and growth of attached coral fragments. This is a surprising and breakthrough observation and describes a new potential for the existing SLIPS principle.

[0018] The design and attachment method to coral fragmentation plugs makes the inventive scheme easily scalable to a wide range of applications. The coated coral plugs formed according to the inventive approach, sometimes referred to as “frag-guards”, can be sold for to the emerging artificial coral reef trade, aquarium trade, and to government organizations to create resilient coastal environments.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 provides an exemplary process flow for fabrication of reef-PDMS and coralguards according to an embodiment of the invention. The lower right panel diagrammatically illustrates a completed coral-guard plug with the layers labeled.

[0021] FIG. 2A is a diagram showing variations in aquarium setups indicating three different pump and water flow arrangements for laboratory reef-PDMS experiments; FIG. 2B diagrammatically illustrates the general setup of the flow-through tanks.

[0022] FIGs. 3A-3B illustrate results of laboratory tests of fouling-prevention coating efficacy on coral plugs, where FIG. 3 A is a bar plot of algal coverage (% of total planar surface area) for control surfaces (coral calcium carbonate plugs) and reef-PDMS plugs exposed to high and low flow treatments after 6 days of incubation of natural seawater (n=12-15 plugs, Tukey HSD post hoc, p<0.01); FIG. 3B provides example images of plugs at 6 days of incubation (letters i-iv correspond to data shown in FIG. 3A).

[0023] FIGs. 4A-4C illustrate aspects of a long term in situ study of fouling-prevention coatings on a coral reef in Hawai'i, where FIG. 4A provides a diagram of an experimental tree for supporting the plugs; FIG. 4B is a photographic image of an algal-covered control plug (left) and reef-PDMS (right) after 12 weeks post-deployment; FIG. 4C plots percentage of algal coverage (means ± SE) on experimental substrates 13 weeks postdeployment (n=20-27). Statistical results are indicated (Means that do not share a letter are statistically significant, p<0.001).

[0024] FIG. 5A illustrates coral growth characterization in coral-guard experiments, where panel l is a top-view image of a coral-guard, showing the slippery transparent ring (sr) and open center (o) for coral fragment attachment; panel 2 provides an image of lateral tissue growth of Stylophora pistilla on coral guard and panel C provides OCT imaging of section shown in panel 2. The cross-sectional OCT image (left) shows the coral tissue (t), the skeleton (s) and a contracted polyp (po). The 3D rendering (right) visualizes the tissue growth front encrusting the coral-guard substrate; FIG. 5B shows growth experiments with attached coral fragments at day 1 of the experiment (panel 1) and after 90 days of cultivation for coral guards (panel 2), PDMS (panel 3) and control substrates (panel 4); FIG. 5C is a violin plot of estimated lateral tissue growth (cm2) after 90 days of cultivation. (ns= nonsignificant).

[0025] FIG. 6A illustrates the effects of coral-guards coral photophysiology, where the upper panel (A) provides typical images of the transient fluorescence (Ft) and the lower panel (B) shows the maximum quantum yield of PS II (Fv / Fm). Imaging was performed such that the laterally growing tissue was in focus. Scale bars = 1 cm; FIG. 6B provides bar plots of Fv / Fm values generated from 15 areas of interest for each of three coral fragments per substrate type.

[0026] FIG. 7 plots algal coverage (%) on control and reef-PDMS surfaces as a function of directional exposure 13 weeks post-deployment at the high flow site. (Data are means ± SD) FIG. 8 is a bar plot of algal coverage (%) on control and reef-PDMS surfaces subjected to high and low flow exposure in situ 5 weeks post-deployment.

[0027] DETAILED DESCRIPTION OF EMBODIMENTS

[0028] The flow diagram in FIG. 1 illustrates steps for fabrication of coral plugs incorporating the inventive SLIPS coating. The i-PDMS SLIPS coatings were applied to coral restoration substrates using standard coral fragmentation plugs made from aragonite sand. Frag plugs are widely available from a variety of commercial sources, including CoralFrags.com and Ocean Wonders. The plugs used for testing were T- or mushroomshaped structures with a circular crown portion having a diameter of about 2-3 cm and a stem portion extending down from the crown. These plugs were selected as the model substrate as they are frequently used in ex situ coral nurseries and in situ coral gardening approaches.

[0029] A common approach for improving PDMS adhesion to glass is through oxygen plasma treatment, however, preliminary experiments on the aragonite plugs suggested that this approach did not noticeably improve attachment. As a result, this step was deemed “optional”. To overcome the adhesion challenge, a simple approach was developed for longterm attachment of PDMS to common aragonite-based coral restoration substrates. A preconditioning protocol (step 100) was developed to reduce leaching of any materials from the plugs (e.g., calcium carbonate), which can interfere with adhesion. Aragonite plugs were dried in a convection oven (Cole Parmer, Model EW-52411-04) at 60°C overnight to eliminate residual moisture. Plugs then underwent sand layer removal to prevent loose sand particles from creating an unstable fabrication base, as preliminary experiments showed that this results in weak adhesion of the PDMS due to rough edges that are prone to tearing and peeling. A 150-grit sandpaper was utilized, and sanding was carried in rotary motion with a circular sanding attachment on a FASTPRO 20V drill. Plugs were then soaked in DI water and shaken vigorously to remove any loose particles, followed by water drainage. The described washing process is repeated three times. Following washing, the plugs are dried again to ensure moisture removal. This protocol ensured a rough and stable aragonite surface, which promotes mechanical interlocking as well as improved bonding and peeling strength of PDMS.

[0030] To prepare the slippery coatings, PDMS-based elastomers were prepared by using the commercially available SYLGARD ™ 184 elastomer kit (Dow Corning, Midland, MI, USA). In step 102, the elastomer base and curing agent were manually mixed at a 10: 1 ratio using a spatula for 10 min. Any bubbles induced during the mixing process were removed by vacuum degassing the mixture for up to 15 min in a desiccator and at a vacuum pressure of 31 mbar. In step 104, the coral plugs were coated with the elastomer. For initial testing, the PDMS was applied by slowly pouring 0.7 gr of the mixture on top of the disk-shaped crown and spin-coated at 800 rpm for 10 s using a spin coater (model SCK-300, Instra Scientific LLC, USA). In an alternative approach, the PDMS (or similar elastomer) may be spray coated onto the upper surface of the plug, with or without spin coating as needed to achieve the desired thickness and uniformity. In step 106, the coated plugs were cured for 48 h in a convection oven (model EW-52411-04, Cole Parmer, USA) at 50°C. Next, the coatings were lubricated for 48 h by complete immersion of the PDMS-coated coral plugs in trimethylsiloxy-terminated PDMS oil with a viscosity of 10 cSt (Gelest, PA, USA- DMS- T11). After lubrication, in step 110, the plugs were tilted at 90° and rotated for 24 h, allowing gravity to aid in removing any excess oil.

[0031] Corals require a stable substrate to support attachment and growth of outplanted fragments. Coral fragmentation plugs made out of materials such as ceramic and calcium carbonate-based sand are widely used as anchoring bases in both ornamental and restoration-focused fragmentation. To reduce the potential for direct competition between macroalgae and coral fragments used in restoration, “coral-guards”, i.e., slippery coral restoration substrates, were fabricated using the process described above and shown in FIG. 1, with additional steps to define an annular shield around the base of the coral. An area at the center of the plug crown was masked to block adhesion of the coating in an attachment target prior to plug coating. For coral-guard fabrication (Step 120), a circular adhesive with a diameter of 0.95 cm was cut from duct tape and attached to mask the center of the preconditioned plug surface (Step 122) to define a placeholder for the coral fragment. Other masking methods may be employed. The base and curing agent of the SYLGARD TM 184 elastomer kit were mixed in a planetary centrifugal mixer (Thinky mixer, Model ARM-310) at a 10: 1 ratio for 210 s at 2000 rpm. (Step 102) While manual mixing was described above, an automated approach enables consistent and homogeneous mixing of larger quantities of SYLGARD 184 in a shorter time without inducing bubbles. 0.9 gr of the premixed elastomer was drop-cast to ensure full coverage of the coral plug disk (Step 104), including the edges to improve adhesion, and cured (Step 106) as described above. A circular stamp (0.95 cm in diameter) was used to define a disk in the cured coating (Step 124). The disk and the mask were then carefully removed using a tweezer (Step 126), leaving an outer PDMS ring with a recessed area at the center to which the coral fragment can be attached. In Step 108, the coatings were lubricated for 48 h by full immersion of the fabricated samples in trimethylsiloxy -terminated PDMS oil with a viscosity of 10 cSt (Gelest, PA, USA) and the excess lubricant was removed in Step 110.

[0032] The lower right panel in FIG. 1 diagrammatically illustrates a completed coral-guard plug, with an enlarged section labeling the different layers.

[0033] Reef-PDMS laboratory experiments. To test the effectiveness of the reef-PDMS coatings in preventing algal biofouling, we evaluated their efficacy in two flow regimes under laboratory conditions. FIG. 2A provides diagrams showing the layouts of flow- through tanks with three variations of the aquarium setup. In each tank, the sample holding, water inlet, and lighting layouts were the same. The only difference is presence or absence, and the number of circulation pumps, during the experiment. The left image shows Variation 1, which had no circulation pump (Reef PDMS). The middle image shows Variation 2, which included 4 circulation pumps on top comers of the aquarium (Reef PDMS exp), directed toward the center of the tank. Variation 3 (right image) included 2 circulation pumps on opposite corners, directed toward the center of the tank (coral-guard exp). FIG. 2B diagrammatically illustrates the general setup of the flow through tanks with 10-gallon flowthrough aquarium system (51.4 cm (L) x 26.7 cm (W) x 32.1 cm (H)). To reduce potential edge effects, the samples were placed at the center of the aquarium, with a minimum distance of 5 cm from the aquarium walls and 10 cm from the water inlet. (Grid mounting height -11 cm adjusted to achieve desired light intensity. Light fixtures were “Orbit Marine 48” IC LOOP PRO Dual LED System w / HUB (4227).

[0034] For the low-flow treatment, flowing seawater was pumped from the SIO (Scripps Institution of Oceanography) pier, and delivered at a flow rate of -100 mL s'1via a 10-gallon flow-through aquarium system (51 *27 x 32 cm). For the high-flow treatment, four circulation pumps (AQUANEAT, 480 GPH, USA) were installed at the four corners of the tank to create a turbulent flow environment. The local flow rate created by the pumps was about -504 mL s'1per pump. For each flow treatment, 12-15 reef-PDMS and control plugs (uncoated), were randomly distributed on plastic crates. Samples were exposed to natural seawater for up to 10 days. During these tests, experimental parameters mimicked coral maintenance conditions.

[0035] Reef-PDMS in situ experiments. The feasibility of the inventive reef-PDMS plugs for in situ applications on Hawaiian coral reefs was tested at two sites with differing flow regimes next to the Hawai'i Institute of Marine Biology (HIMB) at Kane’ohe Bay Bay, O’ahu, Hawai'i. These sites were chosen for testing the coatings under environmental conditions relevant to coral cultivation and restoration. These conditions include a low-flow protected coral nursery (21°25'58"N 157°47'24"W) and a high-flow, exposed reef environment (21°26'15"N 157°47'25"W). The Reef-PDMS plugs were attached to custom- made coral trees (FIG. 4A), which are simple PVC structures fabricated from commercially- available pipe commonly used in outplanting efforts. Each “branch” of the tree was configured to support multiple plugs arranged at approximately uniform angles, e.g., 0°, 90°, 180° and 270°, along multiple cross-sections spaced apart along the length of the branch. The coral trees, which were attached to a pier piling or other support structure facilitated a multidirectional sample placement in order to test the effectiveness of reef- PDMS in response to variations in irradiance, flow regime, and sedimentation effects, thus mimicking natural variations within a coral reef. At each study site, two coral trees were placed with a total number of 25 reef-PDMS and 30 control plugs per structure.

[0036] Coral-guard experiments. The efficacy of the coral-guards was tested compared to coral plug controls (uncoated aragonite-based plugs) along with PDMS-coated plugs that were not infused with the lubricant. Non-lubricated PDMS-coated plugs were tested to assess potential changes in coral physiology that might occur as the result of contact with the lubricant in the coral-guards. For each replicate, a single coral fragment of Stylophora pistillata was glued to the center of the plug using cyanoacrylate- based aquarium glue (RA Aqua Tech, USA). (See. e.g., FIG 5B, panel 1.) Experiments were performed using the flow-through set-up shown in FIGs. 2A-2B with seven replicates for each treatment.

[0037] Coral cultivation. Colonies of Stylophora pistillata obtained from Birch Aquarium at SIO (San Diego, USA) were cultivated in a flow-through aquarium. The seawater temperature in the aquarium was maintained at 25°C, and colonies were kept under a 10: 14 h light-dark cycle, with an incident downwelling irradiance of 100 pmol photons m'2s'1as provided by aquarium lights (Orbit Marine LED Current Loop, USA). Corals were fragmented to 2.5-4 cm in length by transversely cutting the tips of the branches for experimental tests.

[0038] Algal coverage analysis. An underwater camera (Olympus TG-6 12 MP) was used to obtain top-view images from surfaces tested for algal coverage / biofilm coverage in flow-through aquarium systems. In situ underwater images were taken using a Canon G15 camera with underwater housing. Adobe lightroom (Adobe Inc, CA,USA) software was used for contrast and exposure adjustment on the images. During the in situ deployment period, a few of the samples experienced cracking or breakage of the aragonite plugs. Additionally, several images obtained underwater did not meet a satisfactory quality for image analysis due to challenges in maintaining comparable exposure underwater and were thus excluded from the analysis (< 13% of images). Algal coverage was characterized as the two-dimensional area covered by macroalgae (primarily epiphytic turf algal communities) and / or visible biofilms using the open software ImageJ (Fiji, USA). For this, manual segmentation was performed to identify areas of coverage.

[0039] 3D photogrammetry. 3D photogrammetry (see, e.g., Ferrari, R., et al., “3D Photogrammetry Quantifies Growth and External Erosion of Individual Coral Colonies and Skeletons”, Sci. Rep 2017, 7 (1), 16737) was used to quantify coral tissue growth in coral -guard experiments. For this study, samples were removed from the aquarium and placed on a motorized turntable (azimuthal rotation= 0-360°. To optimize contrast and lighting, illumination was provided by an LED panel (Neewer, 20W) at a color temperature of 4000K in front of a white background, and photos were taken with an Olympus TG6 12MP digital camera. Each replicate surface was photographed over two zenith angles (45° and 30°) to yield up to 100 photos per sample taken from different azimuthal angles. A commercial software (Agisoft Metashape Professional, v2.0.3 2023) was used to generate 3D point clouds from the 2D images. Low-quality images were removed, and the remaining photos were aligned with a key point limit set to 0, and a tie point limit set to 10,000. After alignment, a dense point cloud was constructed with accuracy set to high and depth filtering set to mild. The point cloud was scaled using the scaling disk captured in the photos and replicated in the point cloud. Following scaling, all points except those belonging to the lateral coral tissue were trimmed away. This trimmed point cloud was then exported to CloudCompare (v2.12.2022), and a mesh was constructed and trimmed using the PoissonRecon plugin with Octree depth set to 12 and output density as SF selected. Finally, this mesh was exported to Meshlab (2022.02), and the 3D surface area was calculated.

[0040] Pulse Amplitude Modulation (PAM) Fluorometry. PAM fluorometry was performed on the laterally growing coral tissue areas to evaluate the maximum quantum yield of photosystem II (Fv / Fm) for different coral fragmentation samples (control, PDMS, coral-guard) used in the coral-guard experiment. Three individual coral fragments per base material type were tested across five regions on the lateral tissue using an Imaging pulse amplitude-modulated chlorophyll a fluorometer (Imaging PAM, mini version; WALZ GmbH, Effeltrich, Germany) that employs a blue measuring light (460 nm). For each measurement, coral fragments were removed from the experimental system and placed in a black acrylic chamber and dark-adapted for 20 min before employing a saturation pulse (using default settings) to Fv / Fm. Areas of interest for Fv / Fmanalysis were limited to laterally growing tissue that was directly in focus and not obstructed by the coral branch itself.

[0041] Optical coherence tomography (OCT) imaging. OCT imaging was used to visualize 3D coral tissue growth and attachment to the underlying substrate. OCT scans were performed using a Thorlabs Ganymede II (Thorlabs, GmbH, Dachau, Germany) spectral domain system. The instrument has an effective focal length of 36 mm, a lens working distance of 25.1 mm and at the described configuration, the instrument creates scans with axial and lateral resolutions of 5.8 and 8 pm, respectively. Coral fragment samples were placed in a cylindrical glass container filled with seawater. The water level was adjusted at the lateral tissue level to facilitate effective focus on the growth of lateral coral tissue on both the fabricated surface and the aragonite-based control.

[0042] Microcomputed tomography. To further evaluate coral skeletal deposition on fouling-prevention coatings, samples were scanned using a Skyscan 1076 pCT scanner (Bruker, Kontich, Belgium). Samples were mounted horizontally and scanned at 18 * 18 x 18 pm voxel size, applying an electrical potential of 50 kVp, a current of 200 pA, 180° in 0.8° steps, and using a 0.5 mm Al filter. The interfacial layer for coral skeletal deposition was visually observed using custom-written codes in Matlab (Mathworks, Natick, MA).

[0043] Statistical analysis

[0044] Two-way analysis of variance (ANOVA), followed by Tukey HSD post hoc tests were performed to evaluate the efficacy of reef-PDMS coatings in reducing algal cover for different flow environments. Effects of coral guards on lateral tissue growth and Fv / Fmwere tested using a one-way ANOVA. All data was tested for normality of distribution and heterogeneity of variances. Data was loglO transformed if necessary. Reef-PDMS effectiveness in reducing algal cover

[0045] Coral restoration efforts are often challenged by the rapid growth of opportunistic biofilms, filamentous turf algae, and other macroalgae on deployed restoration substrates. This rapid growth results in direct competition with outplanted corals and can additionally have secondary effects on important ecosystem parameters such as reef oxygenation. Coral restoration is often performed in areas that are highly eutrophic and overfished and are thus prone to enhanced growth of coral competitors. To accelerate ecosystem recovery and provide new means for ecosystem engineering, the inventive scheme employed fouling-prevention coatings for coral restoration substrates. As seen in FIGs. 3A and 3B, short-term laboratory experiments revealed the effectiveness of reef-PDMS coatings in reducing early-stage biofilm and algal coverage compared to control coral plugs. For experiments performed under low-flow (described above with reference to FIGs. 2A- 2B), reef-PDMS coatings showed an approximate 2.5-fold reduction in algal coverage compared to uncoated controls (34% ± 12.5% SD vs 84% ± 6% SD, respectively, Tukey HSD post hoc test, p<0.001). For experiments performed under high-flow, this effect was substantially enhanced and reef-PDMS reduced algal coverage >7-fold compared to uncoated controls (12%± 7% SD vs, 92% ± 3% SD, Tukey HSD post hoc, p<0.001). The lower coverage observed for reef-PDMS in the high-flow treatment compared to the low- flow treatment can be attributed to the fouling control mechanism of i-PDMS. The inert nature of PDMS combined with the presence of lubricant oil at the interface of aragonite SLIPS and seawater creates unfavorable conditions for strong adhesion of algal spores. Even though soft fouling assemblages such as algal biofilms may form on SLIPS materials over time, they cannot strongly adhere to the substrate. As a result, increased drag forces present in the high-flow environment are effective in displacing the loosely attached material and organisms from the substrate and disrupt biofilm / algal turf formation and growth.

[0046] To test the long-term in situ efficacy of reef-PDMS on coral reefs in Hawai'i, coral trees (FIG. 4A) with control and reef-PDMS plugs were deployed in two contrasting reef environments (a sheltered coral nursery and open ocean environment) for over 3 months. Results show that the reef-PDMS samples were highly effective in reducing competitive turf algae cover (FIG. 4B-4C). After 13 weeks of deployment, the surfaces of the control plugs were entirely covered with filamentous turf algae and other algal biofilms, while reef-PDMS showed a mean algal coverage of about 30% (± 29 % SD) and 24% (± 19% SD) at low and high-flow sites, respectively (ANOVA, Tukey HSD post hoc, p<0.001, FIG. 4C). Reef- PDMS were effective in reducing algal coverage, independent of directional exposure (FIG. 7) and thus small-scale variations in irradiance and local hydrodynamics. Analysis during earlier timepoints show that after about 3-5 weeks most of the control surfaces are fully covered with algae (FIG. 8).

[0047] Together, these results suggest that such non-toxic fouling-prevention coatings could be an ideal solution to reduce unwanted competitors, such as rapidly growing turf algae, in reef restoration and engineering projects, especially in the early stages of postdeployment. Rapidly growing turf algae can negatively impact ecosystem engineering approaches by producing chemicals and metabolites that deter coral settlement and increase the labile pool of dissolved organic carbon, thereby promoting reef microbialization, unfavorable microbial activity and reef hypoxia. Thus, reef-PDMS coatings could provide a means to affect the temporal dynamics of reef succession in artificial or hybrid reef projects, giving corals a more beneficial macroenvironment as well as biochemical and microbial landscape.

[0048] Mitigation of algal competition via coral-guards

[0049] A core challenge in coral restoration projects is the intense direct space competition of algae with asexually generated coral fragments in coral nurseries and when outplanted onto a degraded reef. Given the successful results of reef-PDMS in reducing competitive algal cover, this mechanism could be leveraged to reduce immediate competition of coral fragments used in coral outplanting efforts. Often, such asexually generated coral fragments are attached to coral plugs and maintained in underwater coral nurseries. Nursery cleaning requires a considerable allocation of the time and resources invested in restoration projects. By creating a protective non-toxic slippery ring surrounding the coral branch, a competition- free environment could be created, thus reducing maintenance efforts. Further, corals should be able to overgrow the slippery coatings given the firm attachment of the central branch, facilitating successive growth over fouling-prevention coatings.

[0050] Lab experiments with the developed prototype coral-guards revealed that corals were indeed able to encrust and overgrow the slippery surfaces (FIG. 5B, panels 2-4). OCT and pCT imaging revealed skeletal deposition directly onto the PDMS- based coating without signs of penetration through the PDMS network. The corals appeared firmly attached to the coral-guards and with no visually-detectable detachment during the 90-day flow-through experiments. This finding demonstrates that despite characteristics such as low wettability and inertness, which make adhesion to i-PDMS based SLIPS unfavorable, coral-guards can support coral tissue growth. This suggests an interesting mechanism by which coral cells could attach and grow on top of a slippery surface, eventually giving them a competitive advantage over opportunistic settlers that are deterred from the substrate.

[0051] Although previous studies support the non-toxic nature of i-PDMS, testing was performed to determine whether there were any potential negative effects on coral growth and tissue health. The results revealed no negative effects on average tissue growth rates on coral guards (5.8 cm2± 2.15 SD) compared to PDMS only (7.6 cm2± 1.78 SD) and control plugs (6.3 cm2± 1.31 SD ANOVA, Tukey HSD post hoc, p= 0.17, FIG. 5C).

[0052] Similarly, Fv / Fmvalues suggested that all corals were healthy, irrespective of treatment and Fv / Fmvalues were on average > 0.6,). As shown in FIG. 6B, there was no significant difference between coral-guards (0.62 ± 0.01 SD) compared to coral plug controls (0.62 ± 0.02 SD) (ANOVA, Tukey HSD post hoc p=0.10), while there was a small difference (mean Fv / Fmdifference = 0.03) between coral guards and PDMS (0.65 ± 0.02 SD, ANOVA, Tukey HSD post hoc, p<0.01). These minor differences in Fv / Fmshould not be a basis for concern due to the susceptibility of the imaging-PAM to suffer from optical artifacts. Overall, these results demonstrate that lubricant infusion and PDMS do not adversely impact coral growth and coral healthiness relative to controls within the scope of the experiments. Accordingly, the inventive coral-guards provide a non-toxic solution to reducing direct space competition with adjacent benthos.

[0053] Global reef degradation urgently calls for innovative, scalable solutions to support current reef restoration and engineering efforts. A major challenge is the rapid settlement and growth of unwanted fouling organisms, such as epiphytic turf algae, which limit coral growth and recruitment. Current leading solutions include mechanical removal of turf algae and the addition of grazers and herbivores.

[0054] According to the embodiments disclosed herein, the bio-inspired SLIPS principle can be applied to develop non-toxic reef-PDMS and coral-guards, which effectively reduce macroalgal cover on reef restoration substrates. These solutions can be easily produced from readily available and well-characterized materials like PDMS. The inventive reef-PDMS provides a sustainable and scalable coating on restoration substrates. Recent studies have also developed sprayable slippery PDMS formulations, enhancing the scalability of this approach while retaining excellent fouling prevention properties. These characteristics make i-PDMS a highly suitable fouling-prevention material for mitigating competitive interactions between corals and macroalgal competitors.

[0055] As described herein, coral-guards have been demonstrated to be highly effective in creating a competition-free environment, facilitating the growth of the branching coral Stylophora pistillita. The inventive scheme is expected to be expandable to other reefbuilding corals with different growth patterns (e.g., mounding, encrusting). The ability to easily modulate the slippery coating facilitates rational pattern design and engineering to enhance coral growth versus competitive algal growth. Similarly, such approaches might be applied to coral recruits or micropropagates that are easily overgrown by adjacent benthos and suffer from low survival rates. The inventive coral-guards will find wide applications in ex situ or in situ coral nurseries.

[0056] The inventive reef-PDMS and coral-guards represent a promising advance in the fields of coral restoration and coral cultivation in marine aquaria. By addressing the issue of unwanted fouling organisms and promoting coral growth, these innovative materials offer a sustainable and scalable solution to support the resilience and recovery of coral reefs in the face of global degradation.

Claims

1. CLAIMS:

1. A structure for reducing competition during coral growth, the structure comprising: a substrate having an upper surface configured for supporting a coral fragment; a coating disposed on at least a portion of the upper surface, the coating comprising a lubricant infused polymer configured to inhibit adhesion of unwanted organisms adjacent to the coral fragment.

2. The structure of claim 1 , wherein the coating has an opening formed therethrough to expose a portion of the upper surface, wherein the coral fragment is attached to the upper surface at the exposed portion.

3. The structure of claim 1, wherein the coating is a poly dimethyl siloxane (PDMS)- based polymer.

4. The structure of claim 1, wherein the substrate comprises a plug having a stem and an upper body, the upper body comprising the upper surface and peripheral edges.

5. The structure of claim 4, wherein the coating is further disposed on the peripheral edges.

6. The structure of claim 1, wherein the plug is formed from a ceramic material or calcium-carbonate.

7. A method of reducing algal competition during coral growth, comprising: providing a substrate configured for supporting a coral fragment, the substrate comprising an upper surface; applying a polymer coating to at least a portion of the upper surface, wherein an attachment area of the upper surface is exposed; applying a lubricant to the polymer coating except for the exposed area; and affixing the coral fragment to the exposed area.

8. The method of claim 7, wherein the polymer coating is a polydimethylsiloxane (PDMS)-based polymer.

9. The method of claim 8, wherein the PDMS is lubricant infused.

10. The method of claim 7, wherein the substrate comprises a plug having a stem and an upper body, and wherein the upper body comprises the upper surface and peripheraledges.

11. The method of claim 10, wherein the coating is further disposed on the peripheral edges.

12. The method of claim 10, wherein the plug is formed from a ceramic material or calcium-carbonate.

13. The method of claim 7, wherein applying a polymer coating comprises: applying a premixed elastomer to the upper surface; curing the premixed elastomer; and removing the cured elastomer from a portion of the upper surface to define the attachment area.

14. The method of claim 7, wherein applying a lubricant comprises immersing the polymer coating in oil for a predetermined period; and removing excess oil.

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