This research was funded by the Ministry of Science and Technology (Taiwan), grant numbers MOST 111-2611-M-291-005 and MOST 111-2811-M-291-001.

1. Hanging coral colonies in&#160;ex situ aquaculture tanks
<ol>
	<li>Position a notched bar (length x width x height: 75 cm x 1 cm x 3 cm), hereafter referred to as a &#34;hanging bar&#34;, across the culture tank in preparation for hanging the coral colonies. 
	NOTE: The hanging bar used in this experiment was custom-made, but a simple PVC pipe with protruding screws (i.e., to act as the notches) would be sufficient as long as it can be positioned in a stable manner across the top of the culture tank and is strong enough to hold the corals.</li>
	<li>Measure a piece of fishing line (see the Table of Materials) to ~1.5 m in length, and then fold it in half twice. 
	NOTE: The initial length of the fishing line should be chosen based on the desired final position of the coral colony in the culture tank.</li>
	<li>Make a small overhand knot at the end of the folded fishing line that has the initial ends of the fishing line. 
	NOTE: After making the knot, there should be two large loops at the bottom and one small loop at the top.</li>
	<li>Place the coral colony in the middle of the two large loops such that the loops are positioned around the colony and can securely hold the coral when it is hung in the water.</li>
	<li>Hook the small top loop of the fishing line into a notch on the hanging bar (Figure 1B).</li>
</ol>
2. Coral feeding
<ol>
	<li>Making the feeding container
	<ol>
		<li>Construct a rectangular frame using acrylic pipe (length x width x height: 25 cm x 60 cm x 25 cm). Make two separate compartments in the frame where the fed and unfed corals can be placed, respectively (Figure 1C). 
		NOTE: Acrylic pipe was used because it is lightweight (i.e., as opposed to heavier PVC pipe) and, therefore, could facilitate easier movement of the feeding container in/out of the culture tanks.</li>
		<li>Use a hot glue gun to adhere 100 &#181;m of plankton mesh to the bottom and sides of the frame.</li>
		<li>Drill a total of ~10 small holes (0.5 cm in diameter) into the pipes (especially along the sides and the bottom of the frame) to prevent the feeding container from floating when placed in the culture tank.</li>
		<li>Drill holes (~0.5 cm in diameter) through the plankton mesh at each corner of the feeding container.</li>
		<li>Place an 8 cm length of 0.5 cm diameter tubing through the corner holes, and use a hot glue gun to fix it into position. 
		NOTE: These pieces of tubing will be connected to an air pump and bubble stones during feeding (see step 2.3.2 for more detail).</li>
	</ol>
	</li>
	<li>Artemia cultivation
	<ol>
		<li>Collect 2 L of seawater from an independent feeding tank, and pour the seawater into an Artemia hatching container (Figure 1D). 
		NOTE: In the present experiment used to demonstrate the protocols, two independent treatment-specific feeding tanks were used, which necessitated the preparation of two hatching containers for Artemia cultivation.</li>
		<li>Connect an air pump to tubing connected to the bottom of the hatching container for approximately 10 min prior to adding Artemia cysts.</li>
		<li>While waiting, use a balance to measure 8 g of Artemia cysts (see the Table of Materials). 
		NOTE: To obtain a mean density of 35 individual Artemia nauplii/mL, as suggested by Huang et al.19, use a ratio of 4 g of Artemia cysts to 1 L of seawater.</li>
		<li>After 10 min, pour the 8 g of Artemia cysts into the hatching container.</li>
		<li>Incubate the Artemia cysts for 48 h.</li>
	</ol>
	</li>
	<li>Preparing the feeding tank
	<ol>
		<li>Place the feeding container into the feeding tank such that the top of the container is above the surface of the water.</li>
		<li>Connect the outer portion of the feeding container&#39;s corner tubing to an air pump, which will supply air to bubble stones for facilitating water circulation during feeding.</li>
		<li>Turn on the air pump ~5 min prior to the start of feeding.</li>
	</ol>
	</li>
	<li>Artemia nauplii enrichment and collection
	<ol>
		<li>Add 1.5 mL of enrichment diet (see the Table of Materials) to the hatching container 2 h before the desired feeding time. 
		NOTE: A ratio of 0.75 mL of enrichment diet to 1 L of seawater is recommended by Huang et al.19.</li>
		<li>After 2 h, turn off the valve supplying air to the hatching container.</li>
		<li>Cover the hatching container with a cardboard box to exclude ambient light, and place a light source (a cell phone flashlight is sufficient) at the base of the hatching container for 5 min to attract Artemia nauplii to the bottom of the container and thereby facilitate the separation of live Artemia nauplii from empty shells.</li>
		<li>After 5 min, remove the box and the light source.</li>
		<li>Place a 3 L measuring jug below the hatching container.</li>
		<li>Detach the tubing from the hatching container to allow the Artemia nauplii and seawater solution to flow into the measuring jug; collect 1 L of the Artemia nauplii and seawater solution. 
		NOTE: Collect only half of the volume in the hatching container to exclude unwanted empty shells.</li>
		<li>While standing in close proximity to the feeding tank, pour the Artemia nauplii and seawater solution through a 100 &#181;m strainer to separate the Artemia nauplii (which will remain in the strainer) from the seawater.</li>
		<li>Rinse the Artemia nauplii held within the strainer twice with water from the feeding tank.</li>
		<li>The Artemia nauplii are now ready to be used.</li>
	</ol>
	</li>
	<li>Feeding the coral colonies
	<ol>
		<li>Unload the Artemia nauplii by placing the strainer from step 2.4.8 into the feeding tank.</li>
		<li>Stir the water in the tank by hand to evenly distribute the Artemia nauplii. 
		NOTE: Collect samples for the &#34;pre-feeding&#34; quantification of the Artemia nauplii density after this step (see step 3.1 for more detail).</li>
		<li>Move each hanging bar (with the coral colonies still hanging from the bar) from the culture tank to the feeding tank, and position the bar so that it is securely resting across the top of the feeding tank. The duration for which the corals are exposed to the air should be kept as short as possible. 
		NOTE: Make sure colonies are not touching each other and have enough space to capture food (e.g., ~5 cm apart).</li>
		<li>Turn off the lights in the feeding tanks, or use a non-airtight lid to cover the feeding tank to avoid light disturbance during feeding.</li>
		<li>Allow the colonies to feed undisturbed for 4 h.</li>
		<li>After 4 h, collect the samples for the &#34;post-feeding&#34; quantification of the Artemia nauplii density (see step 3.1 for more detail).</li>
	</ol>
	</li>
	<li>Post-feeding clean up
	<ol>
		<li>After the feeding session is complete, remove the coral colonies. Take the hanging bars out of the feeding tank individually, and thoroughly rinse each coral with seawater from its respective culture tank to remove any residual Artemia nauplii. 
		NOTE: Rinse the colonies on a stable surface rather than while hanging to reduce the risk of damage that could occur if the colonies were to swing back and forth during rinsing. As per the initial transfer, keep the duration for which the corals are exposed to the air as short as possible.</li>
		<li>Place the hanging bars (with corals hung) back into the culture tanks.</li>
		<li>Detach the tubes connecting the feeding container to the air pump, and remove the feeding container from the feeding tank.</li>
		<li>Rinse the feeding container thoroughly with fresh water to remove all the remaining Artemia nauplii.</li>
	</ol>
	</li>
</ol>
3. Quantifying Artemia nauplii density pre- and post-feeding
<ol>
	<li>Collecting the samples
	<ol>
		<li>Collect samples at two time points: first, when&#160;the Artemia nauplii have been unloaded and distributed evenly in the feeding container (step 2.5.2), and again after the feeding session has been completed (step 2.5.6).</li>
		<li>For each time point, use three syringes to draw 20 mL of water from the surface, the middle layer, and the bottom layer of the feeding container, respectively.</li>
	</ol>
	</li>
	<li>Sample dilution
	<ol>
		<li>For each syringe, transfer the 20 mL of water sample into an independent 500 mL beaker.</li>
		<li>Add 180 mL of hot water (~60 &#176;C) to the beaker (1:10 dilution). 
		NOTE: The hot water is used to immobilize the Artemia nauplii to increase the accuracy of enumeration.</li>
		<li>Add 2 mL of the water sample from the beaker into each well of a 9-well plate. 
		NOTE: Mix the sample in the beaker to distribute the Artemia nauplii evenly in the water column before drawing the 2 mL of sample.</li>
		<li>Count the number of Artemia nauplii in each well under a stereo microscope using 6.5x magnification (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Calculating the density of Artemia nauplii
	<ol>
		<li>Divide the number of Artemia nauplii in each well by 2 to obtain the number of Artemia nauplii per mL. Then, multiply that number by 10 (to account for dilution) to calculate the Artemia nauplii density.</li>
		<li>Calculate the mean density of Artemia nauplii (i.e., average density across the 27 well replicates before vs. after feeding) to compare the Artemia nauplii density between pre- and post-feeding.</li>
	</ol>
	</li>
</ol>
4. Coral larvae collection
<ol>
	<li>Making the larvae collection container (Figure 1E)
	<ol>
		<li>Select a 6 L plastic water bottle, and cut the bottom of the bottle off completely. 
		NOTE: This opening will be used for transferring the colonies in and out of the larvae collection container.</li>
		<li>Create two windows by cutting out a ~15 cm x 20 cm rectangle from each side of the bottle. 
		NOTE: A 6 L plastic water bottle is appropriate for corals that are ~15 cm in diameter; modify the size of the bottle based on the size of the corals being studied.</li>
		<li>Use a hot glue gun and then epoxy to adhere a 100 &#181;m plankton mesh onto each of the windows.</li>
		<li>Create two small holes (~0.5 cm in diameter) on each side of the bottom of the bottle.</li>
		<li>Put a string through the two small holes, and tie both ends to create a handle for hooking the larvae collection container onto the hanging bar.</li>
		<li>Before initial use, place the bottles into a flow-through tank (with no corals) for at least 24 h to remove any glue residue.</li>
	</ol>
	</li>
	<li>Preparing for coral collection
	<ol>
		<li>Immerse the larvae collection container completely into the culture tank.</li>
		<li>Place the colony into the larvae collection container while keeping both the colony and the container submerged in water.</li>
		<li>Hook the handle of the larvae collection container onto the hanging bar. 
		NOTE: After hanging, ensure that the top of the collection container is ~3 cm above the water.</li>
		<li>Repeat steps 4.2.1-4.2.3 until all the colonies are in their larvae collection containers.</li>
	</ol>
	</li>
	<li>Collecting and enumerating the coral larvae
	<ol>
		<li>Prepare a 3 L measuring jug, a bowl, a 3 mL pipette, and 50 mL tubes.</li>
		<li>Unhook the fishing line from the hanging bar, and remove one colony from its larvae collection container. Place the colony back into the culture tank immediately. 
		NOTE: Ensure that the duration of air exposure is as short as possible.</li>
		<li>Place one hand on the cap end of the larvae collection container. 
		NOTE: When the larvae collection container is filled with water, it can be heavy. Without proper support, the container can break when it is being removed from the water.</li>
		<li>Unhook the larvae collection container &#34;handle&#34; from the hanging bar.</li>
		<li>Slowly lift the larvae collection container out of the water.</li>
		<li>Hold the collection container at an approximately 45&#176; angle above the culture tank for a few seconds to allow excess water to flow back into the tank via the larvae collection container windows. 
		NOTE: Do not angle the container past 45&#176; to mitigate the chance of pouring out larvae from the top of the container.</li>
		<li>Remove the larvae collection container from the tank, and position it on top of the measuring jug.</li>
		<li>Before unscrewing the cap, use one finger to apply a moderate amount of pressure against the cap, and then unscrew the cap. 
		NOTE: Water inside of the collection container can be released quickly when the cap is removed if it is not first supported by one&#39;s finger (i.e., potentially resulting in a loss of larvae).</li>
		<li>Transfer some of the water inside of the measuring jug into a bowl.</li>
		<li>Manually count the number of larvae in the bowl by using a 3 mL pipette to move the larvae into a 50 mL tube. 
		NOTE: Be aware that some of the larvae may get stuck inside the pipette. If this happens, draw some seawater into the pipette, and shake gently while sealing the pipette with one finger to loosen the larvae.</li>
		<li>Continue step 4.3.9 and step 4.3.10 until all the larvae have been counted. At this stage, the larvae can be used in subsequent experiments.</li>
		<li>Repeat steps 4.3.2-4.3.10 for all the other coral colonies. 
		NOTE: The measuring jug and bowl should be rinsed between colonies.</li>
		<li>After the counting is finished, rinse each collection container thoroughly with fresh water, especially the windows.</li>
	</ol>
	</li>
</ol>

Ex Situ Culture of &lt;em&gt;Pocillopora acuta&lt;/em&gt; and Effective Feeding Techniques

The authors have no competing financial interests or other conflicts of interest.

This preliminary assessment of the effect of temperature and feeding on coral reproduction revealed differences in reproductive output and timing among colonies cultured under distinct treatment conditions. Further, it was found that feeding Artemia nauplii to coral colonies appeared to be effective at relatively cool (24&#176;C) as well as warm temperatures (28 &#176;C). These combined findings highlight the applicability of these straightforward techniques for the feeding and culture of reproducing scleractini...

Many coral reef ecosystems globally are being lost and degraded as a result of high-temperature stress driven by climate change1,2. Coral bleaching (i.e., the breakdown of the coral-algal symbiosis3) was considered relatively rare in the past4 but is now occurring more frequently5, with annual bleaching expected to occur in many regions by&#160;mid to late century6,7. This shortening of the interim period between bleaching events can limit the capacity for reef resilience8. The direct impacts of high-temperature stress on coral colonies (e.g., tissue damage9; energy depletion10) are intrinsically linked to indirect impacts at the reef-scale level, of which a reduction in reproductive/recruitment capacity is of particular concern11. This has spurred a range of applied research exploring, for example, the active in situ enhancement of recruitment (e.g., reef seeding12), new technologies for scaling-up coral restoration13, and the simulation of reproductive cues to induce reproduction in ex situ systems14. Complementary to these active interventions are the recent recognition of the advantages of heterotrophic feeding in corals under high-temperature stress15 and the exploration of the role that food provision may play in reproduction16.
Heterotrophic feeding is known to influence the performance of corals17 and has been specifically linked to increased coral growth18,19, as well as thermal resistance and resilience20,21. Yet, the benefits of heterotrophy are not ubiquitous among coral species22 and can differ based on the type of food being consumed23, as well as the level of light exposure24. In the context of coral reproduction, heterotrophic feeding has shown variable results, with observations of higher25 as well as lower26 reproductive capacity following heterotrophic feeding being reported. The influence of heterotrophic feeding on coral reproduction across a spectrum of temperatures is rarely assessed, yet in the temperate coral Cladocora caespitosa, heterotrophy was found to be more important for reproduction under lower temperature conditions27. A better understanding of the role of temperature and feeding on reproductive output is likely needed to determine whether specific reefs (e.g., reefs associated with high food availability28) possess a higher capacity for recruitment under climate change.
Similar to reproductive output, the effect of temperature and feeding on reproductive timing in corals remains relatively understudied, despite the synchronization of reproduction with abiotic/biotic conditions being an important consideration for recruitment success in a warming ocean29. Warmer temperatures have been shown to result in earlier reproduction in coral thermal conditioning studies conducted in the lab30, and this has also been observed in corals collected from natural reefs across seasons31. Yet, interestingly the opposite trend was recently observed in fed corals cultured over the course of 1 year in an ex situ flow-through system (i.e., reproduction occurred earlier in the lunar cycle at cooler winter temperatures and later in the lunar cycle at warmer summer temperatures)32. This contrasting result suggests that reproductive timing may stray from typical patterns under conditions associated with abundant energetic resources.
Long-term controlled experiments under different temperature scenarios could contribute to a better understanding of the influence of heterotrophy on reproduction in scleractinian corals. Maintaining reproducing coral colonies under ex situ conditions for multiple reproductive cycles, however, can be challenging (but see previous research32,33). Herein, straightforward and effective techniques for the active feeding (food source: Artemia nauplii) and long-term culture of a brooding coral (Pocillopora acuta) in a flow-through aquaculture system are described; yet, it should be noted that all the techniques described can also be used in recirculating aquaculture systems. To demonstrate these techniques, a preliminary comparison of the reproductive output and timing of coral colonies held at 24 &#176;C and 28 &#176;C under &#34;fed&#34; and &#34;unfed&#34; treatments was conducted. These temperatures were chosen to approximate seawater temperatures in winter and summer, respectively, in southern Taiwan30,34; a higher temperature was not chosen because the promotion of long-term ex situ culture, rather than testing coral response to thermal stress, was a primary goal of this experiment. Further, the density of Artemia nauplii before and after the feeding sessions was quantified&#160;to compare the feasibility of heterotrophic feeding at both temperature treatments.
Specifically, 24 colonies of P. acuta (mean total linear extension &#177; standard deviation: 21.3 cm &#177; 2.8 cm) were obtained from flow-through tanks at the research facilities of the National Museum of Marine Biology &#38; Aquarium, southern Taiwan. Pocillopora&#8203;&#160;acuta is a common coral species that possesses both a broadcast spawning, but&#160;typically brooding reproductive strategy35,36. The parent colonies of these corals were originally collected from the Outlet reef (21.931&#176;E, 120.745&#176;N) approximately 2 years earlier for another experiment32. Consequently, the coral colonies used in the present experiment had been reared for their entire lives under ex situ culture conditions; specifically, the colonies were exposed to ambient temperature and a 12 h:12 h light: dark cycle at 250 &#181;mol quanta m&#8722;2&#183;s&#8722;1 and were fed Artemia nauplii twice per week. We recognize that this long-term ex situ culture could have affected how the colonies responded to the treatment conditions in this experiment. We, therefore, would like to emphasize that the primary aim here is to illustrate how the described techniques can be effectively used to culture corals ex situ by demonstrating an applied example wherein the effects of temperature and feeding on coral reproduction were assessed.
Coral colonies were evenly distributed across six flow-through system culture tanks (tank interior length x width x height: 175 cm x 62 cm x 72 cm; tank light regime: 12 h:12h light:dark cycle at 250 &#181;mol quanta m&#8722;2&#183;s&#8722;1) (Figure 1A). The temperature in three of the tanks was set at 28 &#176;C, and the temperature in the other three tanks was set at 24 &#176;C; each tank had a logger that recorded the temperature every 10 min (see the Table of Materials). The temperature was independently controlled in each tank using chillers and heaters, and water circulation was maintained using flow motors (see the Table of Materials). Half of the colonies in each tank (n = 2 colonies/tank) were fed Artemia nauplii twice per week, while the other colonies were not fed. Each feeding session was 4 h in duration and was conducted in two independent temperature-specific feeding tanks. During feeding, all the colonies were moved into the feeding tanks, including the unfed colonies, to standardize the potential stress effect of moving the colonies between the tanks. The colonies in the fed and unfed treatments were positioned in their own compartment using a meshed frame within the temperature-specific feeding tanks so that only the colonies in the fed condition received food. The coral reproductive output and timing were assessed for each colony daily at 09:00 A.M. by counting the number of larvae that had been released into the larval collection containers overnight.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Artemia cysts&#160;</td>
			<td>Supreme plus</td>
			<td>NA</td>
			<td>Food source&#160;</td>
		</tr>
		<tr>
			<td>Chiller</td>
			<td>Resun</td>
			<td>CL650</td>
			<td>To cool down water temperature if needed</td>
		</tr>
		<tr>
			<td>Conductivity portable meter</td>
			<td>WTW</td>
			<td>Cond 3110</td>
			<td>To measure salinity</td>
		</tr>
		<tr>
			<td>Enrichment diets</td>
			<td>Omega</td>
			<td>NA</td>
			<td>Used in Artemia cultivation</td>
		</tr>
		<tr>
			<td>Fishing line</td>
			<td>Super</td>
			<td>Nylon monofilament</td>
			<td>To hang the coral colonies</td>
		</tr>
		<tr>
			<td>Flow motors</td>
			<td>Maxspect</td>
			<td>GP03</td>
			<td>To create water flow</td>
		</tr>
		<tr>
			<td>Heater 350 W</td>
			<td>ISTA</td>
			<td>NA</td>
			<td>Heaters used in tanks</td>
		</tr>
		<tr>
			<td>HOBO pendant temperature logger</td>
			<td>Onset Computer</td>
			<td>UA-002-08</td>
			<td>To record water temperature</td>
		</tr>
		<tr>
			<td>LED lights</td>
			<td>Mean Well</td>
			<td>FTS: HLG-185H-36B</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Light portable meter</td>
			<td>LI-COR</td>
			<td>LI-250A</td>
			<td>Device used with light sensor to measure light intensity in PAR</td>
		</tr>
		<tr>
			<td>Light sensor</td>
			<td>LI-COR</td>
			<td>LI-193SA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Plankton net 100 &#181;m mesh size</td>
			<td>Omega</td>
			<td>NA</td>
			<td>To collect larvae and artemia&#160;</td>
		</tr>
		<tr>
			<td>Primary pump 6000 L/H</td>
			<td>Mr. Aqua</td>
			<td>BP6000</td>
			<td>To draw water from tanks into chiller</td>
		</tr>
		<tr>
			<td>Propeller-type current meter</td>
			<td>KENEK</td>
			<td>GR20</td>
			<td>Device used with propeller-type detector to measure flow rate</td>
		</tr>
		<tr>
			<td>Propeller-type detector</td>
			<td>KENEK</td>
			<td>GR3T-2-20N</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C&#160;</td>
			<td>To count the number of artemia&#160;</td>
		</tr>
		<tr>
			<td>Temperature controller 1000 W</td>
			<td>Rep Park</td>
			<td>O-RP-SDP-1</td>
			<td>To set and maintain water temperature</td>
		</tr>
	</tbody>
</table>

effective techniques for the feeding and ex situ culture of a brooding scleractinian coral, pocillopora acuta

Climate change is affecting the survival, growth, and recruitment of corals globally, with large-scale shifts in abundance and community composition expected in reef ecosystems over the next several decades. Recognition of this reef degradation has prompted a range of novel research- and restoration-based active interventions. Ex situ aquaculture can play a supporting role through the establishment of robust coral culture protocols (e.g., to improve health and reproduction in long-term experiments) and through the provision of a consistent broodstock supply (e.g., for use in restoration projects). Here, simple techniques for the feeding and ex situ culture of brooding scleractinian corals are outlined using the common and well-studied coral, Pocillopora acuta, as an example. To demonstrate this approach, coral colonies were exposed to different temperatures (24 &#176;C vs. 28 &#176;C) and feeding treatments (fed vs. unfed) and the reproductive output and timing, as well as the feasibility of feeding Artemia nauplii to corals at both temperatures, was compared. Reproductive output showed high variation across colonies, with differing trends observed between the temperature treatments; at 24 &#176;C, fed colonies produced more larvae than unfed colonies, but the opposite was found in colonies cultured at 28 &#176;C. All colonies reproduced before the full moon, and differences in reproductive timing were only found between unfed colonies in the 28 &#176;C treatment and fed colonies in the 24 &#176;C treatment (mean lunar day of reproduction &#177; standard deviation: 6.5 &#177; 2.5 and 11.1 &#177; 2.6, respectively). The coral colonies fed efficiently on Artemia nauplii at both treatment temperatures. These proposed feeding and culture techniques focus on the reduction of coral stress and the promotion of reproductive longevity in a cost-effective and customizable manner, with versatile applicability in both flow-through and recirculating aquaculture systems.

Author Spotlight: Advancing Coral Research by Exploring Climate Change Resistance, Ex Situ Aquaculture, and Reproduction Strategies 

Effective Techniques for the Feeding and Ex Situ Culture of a Brooding Scleractinian Coral, Pocillopora acuta

Our lab is interested in understanding climate change, resistance, and resilience in coral and exploring ex-situ aquaculture's role. Specifically, we aim to cultivate coral in a lab that can be used for restoration and research efforts. Over the past few years, we have seen major advances in coral ex-situ aquaculture, including being able to induce reproduction, as well as complete the entire coral life cycle in both broadcast spawning and brooding corals.Maintaining the long-term reproduction of brooding corals in the lab is usually quite challenging, with reproduction typically stopping after a few months. However, we found that if we culture the colonies together in mesocosm tanks and provide them with supplemental food, they can continue to produce larvae each month. In general, we have a good understanding of how feeding and temperature independently affect coral reproduction, but their interactive effects remain relatively understudied.For example, heterotrophy may have different benefits, or limitations at warmer compared to cooler temperatures. Using the protocols that we have described, we can effectively feed and culture the colonies long-term in X2 aquaculture systems, and can ultimately track the reproduction at the colony level to help infer the influence of heterotrophy on reproduction, and recruitment in a warming ocean.

The described protocols allowed for (1) the comparison of the reproductive output and timing of individual coral colonies among distinct feeding and temperature treatments and (2) an assessment of the feasibility of Artemia nauplii feeding at different temperatures. Herein, a brief overview of the findings is given, but caution should be exercised with regard to the broad interpretation of the reported effects of temperature and feeding on coral reproduction due to the short-term nature of this experiment (i.e.,...

Watch this Scientific Journal Video about Advancing Coral Research by Exploring Climate Change Resistance, Ex Situ Aquaculture, and Reproduction Strategies at JoVE.com

Climate change is impacting coral reef ecosystems globally. Corals sourced from ex situ aquaculture systems can help support restoration and research efforts. Herein, feeding and coral culture techniques that can be used to promote the long-term maintenance of brooding scleractinian corals ex situ are outlined.

Climate change is affecting the survival, growth, and recruitment of corals globally, with large-scale shifts in abundance and community composition ...

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865 Views.  National Museum of Marine Biology & Aquarium. Our lab is interested in understanding climate change, resistance, and resilience in coral and exploring ex-situ aquaculture's role. Specifically, we aim to cultivate coral in a lab that can be used for restoration and research efforts. Over the past few years, we have seen major advances in coral ex-situ aquaculture, including being able to induce reproduction, as well as complete the entire coral life cycle in both broadcast spawning and brooding corals.Maintaining the long-term repr...

Video: Author Spotlight: Advancing Coral Research by Exploring Climate Change Resistance, Ex Situ Aquaculture, and Reproduction Strategies 

Hanging Pocillopora acuta Colonies in Ex Situ Aquaculture Tanks

Begin by positioning a notched bar, also called a hanging bar, across the culture tank for hanging the coral colonies in place. Put the coral colony between the two large loops, ensuring the loops are positioned securely around the colony for stable hanging in water. Hook the smaller top loop of the fishing line into a notch on the hanging bar.

Feeding Pocillopora acuta with Artemia Nauplii

Feeding  Pocillopora acuta with  Artemia Nauplii  

Collect two liters of seawater from an independent feeding tank and pour it into an Artemia hatching container. Next, connect an air pump to the tubing connected to the bottom of the hatching container, and let it run for approximately 10 minutes before adding the Artemia cysts. While waiting, measure eight grams of Artemia cysts using a weighing balance.After 10 minutes, pour the measured Artemia cysts into the hatching container. Next, prepare the feeding tank by placing the feeding container into it, ensuring the top remains above the water surface. Connect the outer portion of the feeding container's corner tubing to an air pump that supplies air to the bubble stones to ensure proper water circulation during feeding.To enrich Artemia Nauplii, add 1.5 milliliters of enrichment diet to the hatching container two hours before the desired feeding time. After two hours, turn off the valve supplying air to the hatching container. After placing a light source at the base of the hatching container, cover it with a cardboard box to exclude ambient light.Allow the light to attract Artemia Nauplii to the bottom of the container for five minutes to separate live Artemia Nauplii from empty shells. After five minutes, remove the box and light source. Place a three liter measuring jug beneath the hatching container.Detach the tubing from the hatching container, allowing the Artemia Nauplii and seawater solution to flow into the jug and collect one liter of the solution within close proximity to the feeding tank. Pour the Artemia Nauplii and seawater solution through a 100 micrometer strainer to separate them from the sea water. Then rinse the Artemia Nauplii inside the strainer twice with water from the feeding tank.Place the strainer into the feeding tank to unload the Artemia Nauplii. Next, stir the water in the tank to evenly distribute the Nauplii throughout the tank. Move each hanging bar with the attached coral colonies from the culture tank to the feeding tank.Ensure the hanging bar is securely positioned across the top of the feeding tank. Remove the coral colonies after the four hour feeding session by taking the hanging bars out of the feeding tank. Thoroughly rinse each coral with sea water from the respective culture tanks.To remove residual Artemia Nauplii, place the hanging bars with the attached corals back into their respective culture tanks.

Quantifying Artemia Nauplii Density Pre- and Post-Feeding of Pocillopora acuta

To quantify Artemia Nauplii used for coral feeding experiments, collect the samples at two time points. At each time point, use three syringes to collect 20 milliliters of water from the feeding container's surface, middle layer, and bottom layer. Transfer the collected water sample into individual 500-milliliter beakers.Next, add 180 milliliters of hot water, approximately 60 degrees Celsius, to the beaker to achieve a 1 to 10 dilution. Add two milliliters of water sample from the beaker into each well of a six-well plate. Next, using a stereo microscope, set to 6.5 times magnification.Count the number of Artemia Nauplii in each well. Density measurements of Artemia Nauplii before and after coral feeding sessions showed that the initial assessment at T0 showed no difference in both temperature treatments. After two and four weeks of culture, the Artemia Nauplii density was lower after feeding in both temperature treatments.There was no difference in the pre-feeding density between temperature treatments, or the post-feeding density between temperature treatments at any of the three time points assessed.

Pocillopora acuta Larvae Collection and Enumeration from Culture Tank

Immerse a larvae collection container completely into a culture tank. Next, submerge the coral colony into the larvae container, ensuring both remain under water. Then hook the handle of the larvae collection container onto the hanging bar.The following morning, unhook the fishing line from the hanging bar, and carefully remove one colony from its larvae collection container. Immediately, place the colony back into the culture tank. Next, remove the larvae collection container from the tank and place it on top of the measuring jug.Apply moderate pressure on the cap using one finger, and unscrew it. Transfer water from the measuring jug into a bowl. Manually, count the larvae in the bowl, using a three-milliliter pipette to transfer them to a 50-milliliter tube.This method enabled the evaluation of the reproductive output of Pocillopora acuta colonies under different temperature and feeding treatments. Colonies cultured at 28 degrees Celsius, released more larvae when unfed than when fed. But the opposite trend was found in colonies cultured at 24 degrees Celsius, whereby the fed colonies produced more larvae than the unfed colonies.Reproduction in all the colonies occurred before the full moon. The mean lunar day of larva release ranged from lunar day 6.5 to lunar day 11.1. However, a significant difference among treatments was only observed between the unfed, 28-degrees-Celsius colonies, which produced earlier in the lunar cycle, and the fed, 24-degrees-Celsius colonies, which produced later in the lunar cycle.