Astrangia spawning, fertilization, and larval development

7/27/22

For the first round of spawning (end of July 2022), corals were collected at Fort Wetherill in the morning by CR and TL. This was also the day of the new moon, but unsure if this has any significance to spawning yet. Corals were in the lab by 12pm

The corals were held in a static tank with a recirculating pump. At first, water temperature was left as is (~24°C), but then a chiller was added to get the temperature to ~22°C.

Corals were glued onto plugs and put into ambient tubs 1 and 2 for acclimation. Temperature in the tanks was around 22-23°C. AI lights were on with UV at 0% and all other colors at 30% (6am sunrise, 6pm sunset, 3 hour ramp time). The tubs were a static system so that if the corals spawned over night or when they weren’t being watched, the eggs and sperm could potentially fertilize in the tub and there would still be larvae!

Once the corals are on plugs and in the tubs, they were carefully watched. If they are happy, their pylops should be out. If they are getting ready to spawn, their polyps may be squat and fat (add photo). The plump/’out’ polyps were poked with a pipette or had water gently squirted on them with a pipette; these usually induced the polyp to spawn.

Astrangia poculata are gonochoric so each polyp will either release sperm or eggs. When the sperm is released, it looks like the polyp is expelling white wispy stuff into the water. When eggs are released, it looks like very small spheres being expelled into the water. Both eggs and sperm enter the water and can be dispersed quickly if pump is present. Spawning can be VERY DIFFICULT to see in these corals so its important to monitor them closely.

When one was actively spawning, it was removed from the tub and put in a glass bowl with just enough FSW to submerge the coral. The coral usually will continue to release gametes over the next few hours. Keep poking the polyps and flushing water on them so they keep spawning. If the coral is releasing sperm, the water in the bowl will become milky and cloudy. Whereas, if the coral is releasing eggs, small eggs will collect at the bottom of the bowl, as the eggs are negatively buoyant. (add pictures)

To facilitate fertilization, 50ml falcon tubes were filled up to 40 ml with 10um FSW. 5mL of concentrated sperm water was added to the tube from one or more spawning males. Then a glass pipette was used to gather lots of eggs and transfer them to the tube. Add picture of pipette with eggs. For this first round, I did 3 glass pipette pulls from bowls with a lot of eggs (add pictures).

After eggs and sperm are in the falcon tubes, lay them horizontally on the benchtop. This will provide more space (?) for fertilization. Invert tubes every 15 mins for 1 hour. After an hour, check out the tube. Depending on density, you might see eggs throughout the tube, but might only see them at the bottom bc they sink but not that big so probably don’t sink fast. To remove the soerm water from the tube pour the tube through a 20um sieve. I prefer this size to the 53um at this step bc it’s easier to see larvae in the tiny sieve mesh. Concentrate the eggs down so that only a little water and eggs are left in the sieve. Transfer the eggs back into the tube with a little water. Eggs should be super concentrated now.

It’s tough to see these coral eggs because they are so damn small. The squaricals were too large a volume of liquid for them - I couldn’t see them and I think they all sank where there was no flow. I put 1/2 of a falcon tube of concentrated down larvae into a glass bowls with around 150ml of FSW in it. The bowls were put in a water bath to bring the bowls up to treatment temperature (22 for ambient, 28 for high).

Because the eggs sink, I used a turkey baster every 2-3 hours to stir them up to optimize development. I would also pipette out some eggs every 4-5 hours and check development under the scope. {add development pictures here}. Some of the bowls had way more eggs than others. I also lost some of the eggs because the water bath spilled over into the bowls and washed the eggs away. My bad.

After about 12-15 hours, I transferred the now larvae into small plastic glad containers with 53 um mesh on the bottom. They didn’t work as well as I hoped, as it was still really hard to see the larvae and I think I lost some when I transferred from the glass bowls to the containers. I capped the containers and let them float in the water bath. It was difficult to keep them upright in the water bath and it was tough to know if they were getting adequate (or any) flow.

After ~24 hours, the larvae had developed in their respective treatments. I sampled from all the containers. To concentrate the larvae down, I poured the water from the containers through a 20um cell strainer. For each container, I sampled the larvae in 1.5ml cryotubes. In each tube, I put 100ul of concentrated larvae. It was difficult to get the same number of larvae for eaach tube, as some containers had way more than others. For each containers, I froze 3 tubes for physiology/molecular/energetics, 1 for size (in zfix), and 1 for molecular (in shield). To reiterate, the number of larvae in each container was incredibly variable.

plan for another spawning night

• for the next round of spawning, I may try something else to let the larvae develop in. I think a small volume of water is important. I may put them in 1L Nalgene bottles with airstones until they are swimming larvae. So I may put several tubes of eggs into a single nalgene and see how it goes. then after they are swimming larvae, maybe put in the squaricals
  • instead of airstones, I’ll use airline tubing to gently bubble in air so that the eggs are kept from fully sinking
• I should also try to do density counts this time around while they are in the nalgene. they will be easier to manipuate
  • need to ask Amy about the specifics
  • Use either the rafter slide or a slide with a scale