Experiments in Coral Sexual Reproduction

1. Introduction to Coral Sexual Reproduction

Although major advances have been made over the last few years, use of sexually-reared corals in restoration is still largely at experimental stage and more costly than asexual propagation techniques. Larval rearing is much more labour intensive compared to asexual techniques, and requires additional expertise, facilities and accurate information on spawning seasonality and timing. There are, however, several reasons for using reared larvae for reef rehabilitation:

• Restoration based on sexual propagation will tend to result in higher genetic diversity than that from asexual propagation.
• Corals are highly fecund, thus coral larval rearing has the potential to produce very large numbers of juvenile corals if the normally high levels of early mortality are reduced.
• Larval rearing should result in little damage to existing reefs, as ‘donor’ colonies can be returned to the wild after spawning.

• Broadcast spawners usually release their eggs and sperm in mass spawning events once a year, governed by environmental factors such as lunar phase and temperature among others. The released gametes drift to the water surface where fertilisation takes place. Commonly after a few days, embryos will have developed into planulae (coral larvae) and settlement will occur.
• Brooding corals, by contrast, reproduce by releasing fully developed larvae instead of gametes (Schmidt-Roach et al., 2012). They have internal fertilisation and embryogenesis before releasing the fully developed larvae. Most of the brooding species release larvae in a temporally cyclic mode, which is triggered by the lunar cycle. They produce fewer larvae than broadcast spawners but they produce their larvae over extended reproductive seasons lasting several months. The time at which corals release their larvae can differ depending on location. Larvae of brooding corals often show less selective settlement behaviour than broadcast spawners. In addition, they generally produce large larvae which already contain zooxanthellae, whereas the broadcast spawners uptake their zooxanthellae shortly after settlement. These factors make brooding corals an ideal subject for laboratory testing.

2. Collection of Larvae from the Coral Colony

There are multiple ways of gathering the larvae from a coral. You can harvest brooded larvae either by placing collecting devices over colonies in situ or by bringing colonies or colony fragments into ex situ aquarium tanks before the predicted time of planulation. For practical reasons, we choose to bring parent colonies into our Fish Lab aquaria, to speed up gathering the larvae and to control environmental variables.

Until now, we have only worked with the coral family Pocilloporidae for our larval rearing, using the following three species: Pocillopora damicornis, P. verrucosa, P. meandrina.

The parent colonies can be collected from the reef by mechanically detaching the colony at the base. When selecting colonies, keep in mind the following: size, depth, health, larvae presence.

• Size – colonies only a few centimetres in diameter may be sexually mature, however, for larval rearing work it is beneficial to use larger colonies where possible. This will increase the number of gametes available for rearing. Ideally each colony or fragment should be 20 to 40 cm in diameter, although for some species (like Pocillopora damicornis) only smaller colonies may be available.
• Depth – coral should be collected from the shallow reef, between a depth of 1 and 5 metres. In this way, the pressure change for the coral is minimal and the colony will be less stressed.
• Health – coral should not have any bleaching spots. Experience shows that if they already show signs of bleaching, the colonies will usually bleach further at an accelerated rate in the aquaria. Therefore, always collect colonies that do not show any sign of bleaching or disease.
• Larvae – ideally, you want to collect a colony that is already fertilised. This way it will be possible to later collect larvae from the colony. If the coral is about to release its larvae, you can see the larvae in the skeleton. Up to three branches should be removed carefully from a colony with scissors or a thin chisel used as a ‘lever’ to snap off the branch. Branches should be snapped approximately halfway between the branch base and tip as this is often where most eggs/larvae are found. The tips of branches and the edges of colonies are often sterile zones and will not provide a reliable indicator of reproductive maturity, however, this method is only reliable when the colony is very close to spawning. If it is unsure whether or not a colony has been fertilised, multiple colonies should be gathered during the collection to spread the chances.
• After collection, the coral can be transported to aquaria for ex situ breeding. The corals should be carefully transported in buckets filled with seawater.

3. Preparation of the Aquarium

Before the collection of parent colonies, the aquarium should be fully prepared. The following factors are crucial for providing the corals with a healthy environment:

• Water flow – parent colonies should be kept in a continuous-flow aquarium, with seawater directly from the ocean to mimic their natural environment. Corals are accustomed to an environment with strong water movement, as the currents bring them food, oxygen, and nutrients, as well as carrying away their waste products. Most corals have little capacity for removing waste material from their surface. In addition, water movement also influences coral growth, growth of algae, and the formation of new coral colonies.
  The parent colonies can benefit from additional water circulation over the entire colony surface by raising colonies above the tank bottom on 5cm pieces piping (to promote water circulation underneath the colony).
• The parent colonies can be placed in a flow-through aquarium equipped with air stones (aeration) and UV light. Light is a central factor for symbiotic corals as it supports photosynthesis. Light endorses the production of photosynthates by the photosynthetic endosymbionts and their translocation to the cnidarian host (Leal et al., 2016). This process largely contributes to coral calcification and growth. Light levels should be similar to the natural habitat; we use Radion XR15w Pro lights to mimic natural light patterns and the lunar cycle.
• Algal should be controlled by introducing grazing organisms to the aquarium such as herbivorous snails, hermit crabs or surgeonfish. We used sea hares in the first month of the coral breeding experiment and this was successful in reducing the algal growth.
  The aquaria should always be kept clean and free from crustose coralline algae (CCA), it is therefore necessary to do at least one full clean of the aquaria per week and to siphon any detritus/sediment halfway through the week. No more than three coral colonies with a diameter of 20 to 40 cm should be kept in an aquarium of 100 litres. It is preferable to keep only a single coral species per aquarium, to be certain of the species of the larvae.
• A temperature meter should be added to one of the aquaria, to monitor the seawater temperature. Normally this will be between 27° and 30° Celsius, with the water temperature being lower in the morning and higher in the afternoon.

4. Collection of Coral Larvae from the Aquarium

Reproductive cycles are often dependent on external cues, such as seasonal temperature and the lunar cycle. Brooders may release larvae daily, without a visible pattern, or have monthly or seasonal reproductive cycles. From experience, we have noticed that Pocillopora tends to start expelling its larvae around sunset. We think the larvae releases peak around full moon and new moon, since we collected most larvae during these periods in the lunar cycle. Larval release, however, could have also peaked as a result of stress. Most peaks were observed around four days after the collection of the coral from the sea.

The larvae can be collected from the parental colonies using different methods:

1. A cylindrical-shaped plankton-mesh capture device can be placed around the colony, so the top of the cylinder is a few centimetres above the water surface. Larvae, which are usually released after sunset (or after the light has been switched off), can then be easily collected from the water surface using a plastic pipette.
2. Shutting off the flow, and preventing the larvae from exiting to the drain. Shutting off the flow, however, can negatively affect the parental colony. When the flow is off for a long period of time the colony will start expelling its polyps and die, so it is important to find a balance. When a colony has not released larvae for a couple of days, it is best for the corals’ health to keep the flow running during the night. Provided colonies are in good health and oxygen levels in the water are adequate, water flow in the tanks should be turned off at around sunset each evening. Later that night, the larvae can be collected and the flow can be turned on again. When the parental colonies are still expelling larvae at night, the flow can be left off and the larvae can be collected the next morning by siphoning via a thin hose.

The larvae can then be transferred either to clean holding tanks or to tanks containing substrates for settlement. For brooded larvae, settlement may begin soon after release, so larvae can be immediately introduced to an aquarium with conditioned settlement substrata.

5. Coral Larvae Tank

For larvae of some common Indo-Pacific species, settlement can occur very soon after release and larvae will settle readily on almost any surface. For this reason, when working with P. damicornis, ensure the holding tanks and settlement tanks are thoroughly cleaned to remove biofilms that will encourage unwanted larval settlement, and to have your conditioned settlement substrates ready as soon as larvae are released.

The substrates that we have used so far are: bare coral rock; coral rock covered in CCA; ceramic tiles covered in CCA.

The coral larvae prefer to settle on substrates that have been ‘conditioned’ in sea water for a period of time. Conditioning broadly refers to the biological succession that occurs on any substrate that is submerged in seawater. Typically, this begins with micro-organisms and is followed by settlement of various types of algae and invertebrates. Corals settle preferentially on substrates that have had time to develop a biofilm and settlement is considerably enhanced when substrate surfaces have some CCA. Conditioning of settlement substrates can be done in the sea or in flow-through aquarium tanks. Pieces of CCA harvested from the reef can be included in conditioning tanks to help speed up the conditioning process. Eight weeks of conditioning has been shown to enhance settlement compared to substrates conditioned for two weeks (Guest, 2010). If other sessile organisms have grown on the substrates during conditioning – for example turf or macro-algae, zoanthids, sponges, bryozoans and ascidians – they should be removed by brushing before coral larvae are allowed to settle.

Besides water quality, water movement and light conditions may play an additional role during the settlement process. We typically use moderate water movement through simple air stones, which increases the chances for larvae–substrate contact, thus enhancing settlement and preventing damage of larvae. The collected larvae should be maintained in clean filtered seawater at ambient temperature at densities of not more than 300 larvae per litre of seawater, with water changes performed at least once each day (flow should be turned off to prevent outflow of larvae). Furthermore, the aquarium should be equipped with an air stone and Radion XR15w Pro lights, which mimic natural light patterns and the lunar cycle. Optimally, the larvae should be kept in ambient temperature to mimic the seawater temperature on the reef (achieved with heaters and water changes).

6. Settlement of Coral Larvae

For brooding larvae, settlement may occur directly after introducing them to settlement substrates. With good conditions, larvae often settle in dense aggregations, which does not represent the most efficient outcome as it can reduce survivorship rates. An appropriate goal is to have a final yield of one surviving coral per substrate unit to be planted onto reefs. The ideal density of settlers starting on each substrate unit to yield a single colony will be around 10 settlers. After enough corals have settled (one to five days after introducing the larvae to the substrate), you need to transfer substrates to a flow-through aquarium for a period of ex situ rearing. Settlers should be left undisturbed for one week to firmly attach and start skeletal deposition. They can then be counted, either using a dissection microscope or by eye with the aid of blue lights (as coral tissues often glow green under fluorescent light, making settlers more perceptible).

The biggest challenge that the larvae will face after successful settlement is algal growth. Algae will smother corals in their first life stages, so control is essential by introducing grazing organisms (repeat the aquarium preparation stage) while not damaging the juvenile corals.

Maintaining the corals in ex situ tanks for a period after settlement can significantly increase survivorship. The longer the coral is reared ex situ, the better the chance of survival. After the first settlement score, larvae can be monitored regularly to observe growth and survival. This can be done by using the dissection microscope.

7. Feeding of Aquarium Corals

The corals that we are breeding are symbiotic corals; for their food they rely on hetro- and autotrophy (photosynthesis and consumption of plankton). The animal host is heterotroph, ingesting a wide range of particles, while the symbionts are autotrophs, through their photosynthetic activity. Photosynthates are, however, very rich in carbon, but often deficient in other elements such as nitrogen and phosphorus. To stimulate growth and health of the parent colonies and the young corals, the corals will need to be fed regularly. Toh et al., (2014) showed in their studies that juvenile specimens of Pocillopora exhibited almost cubic increases in growth and survival when they were fed twice a week with Artemia saline. Only the settled corals should be fed, since the planular larvae will not start feeding until they are settled and metamorphosed. In our Fish Lab, settled larvae and juvenile corals were fed Rotifers, whereas the parent colonies and juvenile corals (older than one month) were fed the larger Artemia.

The Rotifers should be cleaned of waste and yeast before feeding them to the corals, using a sieve (50um mesh) and seawater. Never allow the Rotifers to dry out (they will quickly die). After rinsing, the Rotifers are enriched with one shot of S-presso for ± 10 minutes with an air stone, then rinsed for a second time. Then they are ready to be fed to the corals.

The Artemia follow a similar process (using a 80um sieve).

After the collection of the food, the flow in the aquarium should be turned off and drained to a level below the water outlet (to prevent loss of the Rotifers/Artemia through the drain). To ensure successful feeding, the coral polyps should be extended, otherwise they will be unable to catch the plankton from the water column. Ten minutes after the addition of the food, the flow should be restarted (slowly) to create water movement which will give the coral more opportunities to catch the food.

8. Weekly Work Schedule

The following tasks are essential during the week:

• Feeding: every other day (3x per week)
• Recording seawater temperature: every day
• Flow on/off when trying to collect larvae: every night
• Collect larvae: every morning
• Water change larvae: every day (when you have larvae)
• Monitor primary polyps: 1x per week
• Deep clean of aquaria

9. Successes in Coral Sexual Reproduction

84 polyps successfully settled, with a survival rate of 29% (25 surviving colonies). These colonies vary in size depending on their positioning on the substrate, with our largest colonies measuring 2cm. The species with the highest success rate is Pocillopora verrucosa.

One year on, colonies are displaying signs of secondary branching (an excellent sign of health) and we hope to transplant them to a coral frame in the near future, where further monitoring will take place.

Coral Text References

• Guest, J. (2010). Rearing coral larvae for reef rehabilitation. Reef rehabilitation manual, 73-92.
• Leal, M. C., Ferrier‐Pagès, C., Petersen, D., & Osinga, R. (2016). Coral aquaculture: applying scientific knowledge to ex situ production. Reviews in Aquaculture, 8(2), 136-153.
• Schmidt-Roach, S., Miller, K. J., Woolsey, E., Gerlach, G., & Baird, A. H. (2012). Broadcast spawning by Pocillopora species on the Great
• Barrier Reef. PLoS One, 7(12), e50847.
• Veron, J. E. N., & Stafford-Smith, M. (2000). Corals of the World. Volumes 1-3. Australian Institute of Marine Science, Townsville, Australia, 1382.
• Toh, T. C., Ng, C. S. L., Peh, J. W. K., Toh, K. B., & Chou, L. M. (2014). Augmenting the post-transplantation growth and survivorship of
• Juvenile scleractinian corals via nutritional enhancement. PloS one, 9(6), e98529.