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Home Protocols Miscellaneous Coral Spawning for Dummies

Coral Spawning for Dummies

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(Acropora palmata and Montastraea faveolata)
Last updated 7/20/09
Estimating spawning dates:
Coral spawning in the Caribbean is highly dependent on the full moon in the late summer months.
Full moon: Full moon predictions are available from multiple sites online:
Acropora palmata: This species has been reported to spawn 3-6 nights (highest probability nights 4-5) after the full moon in late summer (mainly August, occasionally in September) in multiple locations throughout the Caribbean (see Table). This species spawns 2 hours after sunset.
Montastraea faveolata: This species seems more predictable in its spawning clock, occurring 5-7 nights (highest probability nights 6-7) after the full moon in late summer (see Table). This species spawns 2.5-3 hours after sunset.
It is best to be checking a bit earlier by sending a diver down. Attach glow sticks for finding tents after sun down.
Preparation for spawning:
Net fabrication: The spawn collectors can vary in size depending on the target colonies. Nets are normally made out of see-through fabric or mesh. Dress lining material and fine-weave sheer curtain materials are the least expensive. They can be tied down to the rubble or have weights at the bottom end that hold them over the colony. Attach a float to top end of net. The cod end must be narrow to funnel the gametes into a collecting jar. Two ways of accomplishing this are suggested. The cod end of the net can be fortified and shaped with a funnel, in which case you can make a hole in the center of the cap of the collecting jar and push the cap down over the pointy end of the funnel. A bottle or jar is screwed onto the cap to capture the spawn.Otherwise, the net can be shaped by the way the material is cut and sewed (like a flared panel skirt), and then the opening can have an elastic or drawstring to hold a collecting jar or bottle in place. make sure the collecting container has some sort of rim on it so that it doesn’t get out from the elastic In either case, the divers must remember to take down caps to close off the containers when they are removed from the net. 
Coolers and wet lab area: Large coolers for the spawn are cleaned and set up prior to the first day of spawning. The wet lab area for larval clean up and transfer should also be ready with necessary supplies (i.e. transfer pipets, miscellaneous plastic containers, gravy boats).
Set up seawater filtration system to have access to enough filtered seawater (FSW) prior to the night of spawning.Have sufficient filtered seawater on hand to rinse the sperm and to fill the culture chambers with when you get back from the field. Water can be filtered through a GF/A filter or a 5 um string filter cartridge for culturing, since you are not trying to achieve sterile culture conditions. Spawn brought back from the field with have plenty of contamination withzooplankton and other critters which will be gradually cleaned out with the water changes.
Set up collectors: The dive and boat teams are assigned prior to the nights of diving. Designated teams (2 divers, 1 snorkeler) set up the spawn collectors before dusk. Make sure you set out a few strategically placed cyalumes to guide the divers during the spawn dive, and also assist with removing nets. You can use different colored cyalumes for different tasks (divers, nets, mooring buoy).
Spawning: The gamete bundles are approximately 1mm in diameter and are positively buoyant. They are concentrated in the bottle that is maintained also in a buoyant position due to a small float located above the bottle. The divers will bring extra lids for the bottles on the nets and seal the container still upside down to keep the spawn in the bottle. The divers hand the spawn to their snorkeling partner who will swim with the bottle back to the boat. Be careful not to lose the lids!!! As soon as a bottle is full, swim back with the spawn to the boat to prevent bundle breakup. This is critical as the fertilization can take place in lower seawater volumes than when the bundles break up and the sperm is more diluted. When checking for spawning, avoid shining dive lights directly on the nets as this will attract miscellaneous planktonic predators.
Fertilization (Alina’s recipe): Normally this happens on the boat as snorkelers bring the containers back from the reef. Guestimate concentration of sperm (slightly milky, not too milky). According to Alina, ½ the whiteness of skim milk appears to be about right for good fertilization. A final sperm concentration of 1-2 x 10^6 sperm per ml is the recommended standard. The volume of unbroken bundles to total volume of water should be ca. 1 part bundles + 4 parts seawater (i.e. bundles make up 20 % of total volume). This becomes more difficult as bundles start to break up. That is when the degree of milkiness seems to work. It is a good idea to take subsamples of the sperm to do counts and figure out the final concentration used in the end.
Since the amount of spawn is unpredictable, it is best to bring multiple plastic graduated containers on the boat to be able to start with a small container for a small mix of gametes. Then transfer to larger containers as more spawn becomes available.
As each container comes to the boat, examine it to get an idea of how full of bundles it is. If there are lots of bundles per size of container, then the amount of extra water in the container will be fine and you can dump the entire contents into your fertilization bucket (e.g. small container, lots of spawn).
If there are few bundles per size of container, then you will NOT want to use all the water in the container [THIS ASSUMES THAT THE BUNDLES HAVE NOT STARTED TO BREAK UP AS OF YET]. In this case, position the container upside down inside a larger container and try to loosen the cap and let a small amount of the water out, and let some air into the container. Then turn the container upright and let the bundles rise to the surface. Open up the container, and pipette out the bundles into the spawn bucket, and discard the extra water in the container. A good way to pipette out the bundle is to use the bottles used by the Knowlton team in Panama. See picture below
If there are few bundles and they’ve already broken up, set container aside to decide later whether you want to use it. If you have tons of spawn then a dilute sperm concentration from such containers won’t be a problem, and adding spawn from more colonies will increase gamete pool diversity. But if you don’t have all that much spawn, try to take the eggs off with a pipette and discard the seawater, or use just a small amount of it to increase odds of fertilization of eggs from other colonies.
Let fertilization take place for about one hour as the boat gets back to the lab. Mix the mixture well after adding each new batch of spawn, and try to keep from splashing up the sides of the container during the trip home. BTW, the sperm concentration in this photo was outrageously high; I’d aim for 1/3 that level of milkiness.
Larval cultures: Upon arrival in the lab rinse the sperm out with FSW until the water is clear (could be 4-5 times). Be very gentle with these changes so as not to damage the eggs. It helps to have the spawn in a container that has a bottom drain (e.g. cooler) so that you can release the dirty water from below, and add clean water from above. Otherwise siphon out the water from below the eggs. DO NOT USE THE BIG SIEVES AT THIS STAGE.  If you are near a scope, wait ca. 1 hr after you wash out sperm and you should see embryos in the 2-3 cell stages. You can assess percent fertilization by counting the percent of eggs not dividing.
Once clean of sperm (try to get out copepods and fish larvae too), transfer the fertilized eggs to the culture coolers or bins also in FSW. Eggs should form a thin layer covering ca. 20 % of the surface area. For the blue cooler size, this is ca. 2 ml of packed eggs for Montastraea and 11 ml of eggs for Acropora. Another way to estimate for other sized containers (goal is ca. 1-2 eggs per ml) is 3 ml of packed eggs per 10 L volume for Acropora and 0.6 ml of packed eggs per 10 L for Montastraea.
The cultures have to be transferred approximately once a day into fresh FSW and the culture containers cleaned as follows: If there is a surface film containing white greasy scum, you can carefully lay a piece of Saran wrap over the surface and carefully lift off. Much of the scum will stick to the film and few embryos will. You can do this several times to help remove much of the dead floating waste materials. It is best to try to either pipette or scoop the as much as possible of the floating embryos from the water surface into containers with clean filtered seawater before doing the next step. Then siphon most of the water out of the coolers with the siphon tube inserted into a large PVC/mesh filter (100 uM for Montastraea and 200 uM for Acropora). When the water volume is small enough, pour into a 500 ml to 1 L container to pick out the rest of the embryos. Then the larvae are successively transferred to smaller containers and drained with smaller PVC filters until the volume can be transferred into the gravy boats for the final cleanup. The larvae should always remain wet by using squirt bottles to rinse them off the walls as the cooler/containers are drained.
While the embryos/larvae are being cleaned, the coolers have to be rinsed with freshwater multiple times and refilled with FSW. The larvae have to be occasionally (ca. 1 hr is ideal) stirred to keep them from sticking to the cooler walls. Sample embryos and inspect under scope for fertilization success. If fertilization is poor will decaying eggs foul it up and necessitate even more frequent cleaning?
            After ca. 2 days for Acropora and 1.5 days for Montastraea, the gravy boats may not work any longer because the larvae stop floating. Then only the siphon tubes and small filters can be used to concentrate them for cleaning. Try to use the Saran film and to pipette off clumps of larvae before you siphon/filter to reduce physical stress of the mesh on the larvae.
After 3-5 days the cultures should be more stable and can be transferred to smaller containers for planned experiments.