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 corals and exploring ex-situ aquaculture's role. Specifically, we aim to cultivate corals 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 lifecycle 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 coral needs together in miso custom 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 ex-situ aquaculture systems, and it can ultimately track the reproduction at the colony level to help infer the influence of heterotrophy on reproduction and recruitment in a warming ocean.

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.

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 mL 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 seawater. 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 seawater from the respective culture tanks to remove residual Artemia nauplii.

Place the hanging bars with the attached corals back into their respective culture tanks. 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 mL of water from the feeding container's surface, middle layer, and bottom layer.

Transfer the collected water sample into individual 500 mL beakers. Next, add 180 mL of hot water, approximately 60 degrees Celsius, to the beaker to achieve a 1:10 dilution. Add two mL 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 T-zero 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. 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 3 mL pipette to transfer them to a 50 mL 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 larval 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.