This post includes the full notebook for the June-July 2023 Montipora capitata coral spawning and field expedition in Hawaii at the Hawaii Institute of Marine Biology. My project will focus on development of M capitata larvae through their use of energetic stores and expression of coding and non-coding transcripts. Dr. Ariana Huffmyer is also conducting experiments on M. capitata larvae; her experiments are detailed in her Github notebook here. Github for the developmental timeseries is here.

20230615

Arrived! Slowly but surely.

20230616

Set-up

Supplies and equipment were unpacked and lab space was set up. AH filled the dry shipper up with liquid nitrogen at UH Manoa to act as a dewar in the field. Incubators were unpacked and set up. Incubators were set to ambient (27°C) and high (30°C) temperature treatments.

Tube prep & labeling

JA prepped 50 mL falcon tubes and 1.5 mL tubes for sampling. The 50 mL falcon tubes will be used to hold the larvae in the incubators. The 1.5 mL tubes will be used for sampling for physiology (carbs, lipids, protein), molecular (mRNA and ncRNA) and size. 1.5 mL tubes were labeled in the following way:

Color	Category	Number Range	Buffer/preservation	Storage	Destination
Blue	Size	S4-S94 (no S16)	20% Z-fix	4C; transport room temp (or on ice packs)	URI
Orange	Molecular	M1-M94 (no M16)	RNA/DNA shield	-80C; transport ln2	URI
Green	Physiology	P1-P94 (no P16)	Snap freeze	-80C; transport lN2	URI

Logger deployment

2 loggers were deployed in each incubator. We found that there was variation in the temperatures on the different shelves of the incubators, with the top shelf being the warmest and the lowest shelf the coolest. The incubators were filled with 50 mL falcon tubes at this time to test airflow. We will need to reduce the number of falcon tubes in the incubators for the developmental time series experiment. Below is the metadata for each logger:

SerialNumber	Type	DateDeployed	Incubator
21723476	HOBO Tidbit MX2203	20230616	1
21723475	HOBO Tidbit MX2203	20230616	1
21723473	HOBO Tidbit MX2203	20230616	2
21723474	HOBO Tidbit MX2203	20230616	2
21723472	HOBO Tidbit MX2203	20230616	3
21723477	HOBO Tidbit MX2203	20230616	4
20987156	HOBO U22	20230617	3
20444042	HOBO U22	20230617	4

Spawning

The CRL team went to Reef 13 in Kaneohe Bay to collect gametes from wildtype spawning slicks. They reported that spawning was light and they were able to collect some gametes for preliminary testing. Two falcon tubes of gametes (5 mL gametes per tube in 40 mL FSW) were used for testing. Tubes incubated on the bench top until all bundles were broken up around 23:00. Tubes were rocked every 10-15 minutes to facilitate bundle breakage. After the majority of bundles were broken up, the fertilized eggs were rinsed 3x with FSW to clean the sperm.

Developmental time series testing

Since spawning was light, AH and JA decided to run a test for the developmental time series experiment to see if the embryos could survive in the 50 mL falcon tubes with minimal water changes. Stocking density was also assessed.

To calculate the concentration of eggs, 5 uL was taken from the concentrated egg stock and counted under the micrscope (average of 76 eggs in 5 uL concentrated stock). Based on this estimate, tubes were stocked with either 10, 50, 100, 200 or 500 uL of the concentrated egg stock (n=3 tubes per density per temperature treatment) in 40-45 mL FSW. Stocking took place from 22:50-23:15. Below is the metadata from the prelim 50 mL falcon tubes:

FalconID	DateLoaded	Volume_uL	Treatment
30	20230616	10	Ambient
43	20230616	10	Ambient
46	20230616	10	Ambient
50	20230616	50	Ambient
41	20230616	50	Ambient
55	20230616	50	Ambient
65	20230616	100	Ambient
154	20230616	100	Ambient
94	20230616	100	Ambient
21	20230616	200	Ambient
176	20230616	200	Ambient
88	20230616	200	Ambient
104	20230616	500	Ambient
191	20230616	500	Ambient
162	20230616	10	High
143	20230616	10	High
109	20230616	10	High
139	20230616	50	High
140	20230616	50	High
142	20230616	50	High
148	20230616	100	High
130	20230616	100	High
170	20230616	100	High
181	20230616	200	High
164	20230616	200	High
105	20230616	200	High
63	20230616	500	High
224	20230616	500	High

20230617

Prelim tube checks

at 03:00:

• Ambient has some clumping in all tubes, but more so in the higher density tubes.
• All in high treatment look like they have some clumping and death. Water is more opaque in these tubes than the ambient tubes. Also less evidence of fertilization in high treatments. Less debris and water is cloudier.
• Rocking tubes every 30-60 minutes will help with clumping. As for high treatment, maybe JA can use a glass pipette to remove debris periodically? Don’t want to do frequent water changes, as I don’t want to disturb them too much

At 08:00:

• Water changes performed on all tubes. Water changes performed by pouring contents through a cell strainer, refilling the tube with fresh FSW and then adding the embryos back in.
• Put filled tubes in middle rack in incubator.
• Changed temps to be set at 26°C for ambient and 29°C for high.
• Calculated out what I’ll need for my actual sampling: 6 time points x 2 treatments x 7 replicates = 84 tubes

Sampling prelim test

JA did a sampling run-through to prep for actual sampling. She decided to use one 50 mL tube to sample for all 3 metrics (physiology, molecular and size). Here’s the sampling protocol:

• Remove a 50 mL tube from the incubator and record the tube ID and time it was removed from the incubator.
• Pour the contents through a cell strainer and set the cell strainer in a 6 well plate with a little bit of FSW.
• Sample 20 uL of the concentrated embryos for size. Preserve in 20% Zfix. Note the time that this sample was preserved. Store at 4°C.
• Use a P1000 to remove the rest of the embryos. Record how much volume total it took to collect the embryos. Put these embryos in a 20 mL glass vial.
• Subset 20 uL of the embryos and count how many embryos are in that volume. Record this number. Using the volume of the embryos, the total number of embryos in the 50 mL falcon tube can be calculated.
• Move half of the total volume to the molecular tube and the other half to the physiology tube. This should split the embryos evenly between the physiology and molecular sample tubes.
• Preserve the molecular tube by adding 500 uL of DNA/RNA shield. Record the time preserved. Store at -80°C.
• Preserve the physiology tube by snap freezing in liquid nitrogen. Record the time preserved. Store at -80°C.
• Sampling complete for one falcon tube! Repeat for all other falcon tubes for that sampling time point.

Spawning (part 2)

Much more spawning tonight! CRL team brought back 6 50 mL falcon tubes (5 mL gametes in 40 mL FSW) at 21:45. Tubes were left on the benchtop to fertilize and rocked gently every 5 minutes. After an hour, the fertilized eggs were rinsed 2x with FSW.

Loading tubes

Decided to load the 50 mL falcon tubes with 200 uL stocking density. The embryos seemed to survive well at this density and it will provide a sufficient number of larvae to sample from each tube. 200 uL of concentrated eggs were loaded into 50 mL tubes with 40-45 FSW. Tubes were inverted gently and loaded into an incubator with the tubes laying on their sides. Incubators were set to either 26°C or 29°C. 24 tubes with 200 uL of concentrated eggs were loaded into each incubator (4 incubators total; 2 ambient, 2 high). 1 tube per incubator was loaded with no eggs to serve as a “blank”.

Incubator	Treatment	Time Tubes Loaded	Tubes in Incubator
1	Ambient	22:50	23:05
2	High	23:39	23:53
3	Ambient	23:05	23:20
4	High	23:25	23:39

Sampling

Sampling of the fertilized egg stock was done at 23:30 for physiology, molecular and size (all at ambient). Sampling was done according to the protocol above.

20230618

Tube upkeep

After being loaded into their respective incubators, tubes were gently rocked every hour to prevent embryos from clumping. This helped reduce die-off and kept the embryos developing at a regular pace. One 1L Nalgene of FSW was kept in each incubator for water changes so that the water was already at the treatment temperatures. Water changes on all tubes were done at 06:30 and 16:45. Water quality measurements (temperature, pH, salinity) was taken at 00:31, 05:45, and 16:20 on 3 tubes per incubator. Water quality measurements for today are below:

Date	Time	FalconID	pH (mV)	pH_nbs	temp (C)	sal (psu)	Tris date	Notes
20230618	0:31	Nalgene1	-52.8	8.08	23.87	33	20230617	Nalgene1 was sitting on bench prior to measurements
20230618	0:34	Nalgene2	-51.1	8.05	28.3	33	20230617
20230618	0:37	Nalgene3	-51.2	8.05	28.51	33	20230617
20230618	0:39	Nalgene4	-52.3	8.07	25.5	33	20230617
20230618	5:46	22	-45.2	7.94	24.6	34	20230617
20230618	5:47	184	-45.5	7.95	24.51	34	20230617
20230618	5:49	69	-47.7	7.99	25	34	20230617
20230618	5:51	81	-46.6	7.97	29.15	34	20230617
20230618	5:53	53	-45	7.94	29.92	34	20230617
20230618	5:54	18	-41.8	7.89	29.53	34	20230617
20230618	16:19	87	-30.4	7.69	28.37	34	20230617
20230618	16:20	75	-6.4	7.27	29.34	34	20230617
20230618	16:22	27	-23.5	7.57	29.25	34	20230617
20230618	16:24	76	-40.2	7.86	28.03	34	20230617
20230618	16:26	51	-38.9	7.84	28.51	34	20230617
20230618	16:28	202	-40.2	7.86	27.51	34	20230617
20230618	16:30	52	-36.8	7.8	25.23	34	20230617
20230618	16:32	70	-28.1	7.65	25.18	34	20230617
20230618	16:33	71	-41.2	7.88	25.33	34	20230617
20230618	16:35	36	-32.3	7.72	25.35	34	20230617
20230618	16:37	Nalgene1	-50.1	8.03	24.61	34	20230617
20230618	16:38	Nalgene2	-47.1	7.98	28	34	20230617
20230618	16:39	Nalgene3	-45.6	7.95	28.8	34	20230617
20230618	16:40	Nalgene4	-47.9	7.99	25.14	34	20230617

Sampling

Sampling took place at several time points today: 4, 9, 14, and 22 hours post fertilization (hpf). Sampling was done as detailed above. At each time point, n=6 falcon tubes were sampled from each treatment (n=3 falcon tubes per incubator). Total embryos were calculated for each tube, but this number is approximate. Some molecular samples (tubes M10, M11, M12, M17 and M18) may not be viable. In these tubes, there was already shield in the tubes when adding the sample.

20230619

Tube upkeep

Tubes were checked every 3-6 hours and rocked gently to prevent build up. Embryos are looking good! JA periodically took an aliquot out of the tubes to check development under the microscope. Water changes occurred at 08:00. Water quality data was also collected:

Date	Time	FalconID	pH (mV)	pH_nbs	temp (C)	sal (psu)	Tris date	Notes
20230619	22:40	14	-45	7.94	26.08	34	20230617
20230619	22:41	39	-35.3	7.78	25.64	34	20230617
20230619	22:42	47	-45.5	7.95	28.59	34	20230617
20230619	22:43	89	-36.2	7.79	28.68	34	20230617
20230619	22:45	44	-43.1	7.91	29.63	34	20230617
20230619	22:46	75	-38.2	7.83	29.54	34	20230617
20230619	22:48	72	47.8	7.99	24.92	34	20230617
20230619	22:49	52	-35.9	7.79	25.52	34	20230617

Sampling

Sampling took place at several time points today: 28 and 48 hpf. Sampling was done as detailed above. At each time point, n=6 falcon tubes were sampled from each treatment (n=3 falcon tubes per incubator). Total embryos were calculated for each tube, but this number is approximate.

20230620

Tube upkeep

Tubes were checked every 3-6 hours and rocked gently to prevent build up. Embryos are looking good! JA periodically took an aliquot out of the tubes to check development under the microscope.

Sampling

Final sampling today!! Occurred at 72 hpf. Sampling was done as detailed above. At each time point, n=6 falcon tubes were sampled from each treatment (n=3 falcon tubes per incubator). Total embryos were calculated for each tube, but this number is approximate.

The following table has the hpf, as well as the start and end times for each sampling time point.

Date	hpf	TimeStart	TimeStop	NumberOfSamples
20230617	Fertilized egg	23:30	23:39	18
20230618	4	2:45	4:20	36
20230618	9	8:41	10:38	36
20230618	14	13:53	15:51	36
20230618	22	20:57	22:54	36
20230619	28	2:15	4:05	36
20230619	48	21:36	23:21	36
20230620	72	21:26	23:25	36

Each time point took me about ~2 hours to sample, so for each sample, its not exactly 4 or 9 hpf. I alternated sampling between high and ambient treatments, so there should be no effect of time on treatment if that makes sense.

Hooray! Sampling for my project complete! See A. Huffmyer’s notebook posts and project github for the other experiments that were completed during this trip.

20230703

Fungia spawning

Did some Fungia spawning with the Hagedorn lab today. 25 adult Fungia colonies were put in 3L containers at 15:45 today with FSW. The containers were in a water table with flow-through FSW. Colonies were monitored for spawning until 18:00, but no spawning occurred.

20230704

Fungia spawning & embryo rearing

Another spawning night attempt with Fungia. Like last night, Fungia colonies were placed in containers in the water table with FSW and monitored for spawning. Only 1 male and 2 females spawned - these gametes were combined in a large bowl for fertilization. After 30 minutes, the sperm water was siphoned through a 40 um mesh sieve and new FSW was added; this was done twice to clean the developing embryos. The embryos were placed in a 3L plastic container and left in the Hagedorn rearing room overnight. The embryos appear to be negatively buoyant and do not have symbionts. When we left them for the night, they were all mostly on the bottom of the container.

20230705

Embryo rearing (from 7/4 spawn)

Embryos were rinsed this morning. First, embryos were scooped up with a small glass pyrex bowl and passed through a 300 um mesh sieve; this was done to collect any larger pieces of debris. The embryos that passed through that sieve were then scooped and put through a 40 um mesh sieve, which collected and concentrated them. The embryos were then put back into their container and the container was refilled with FSW. Hagedorn lab said this only needs to be done once a day.

Fungia spawning & embryo rearing

Big spawn tonight! 9 males and 8 females spawned in the water table around 18:30. The Hagedorn lab collected sperm and eggs for their cryopreservation and experiments, and the rest was left to fertilize for 30 minutes. After fertilization, the embryos were rinsed 2x with FSW (similar methods to 7/3). The embryos were then put in 3L containers and kept overnight in the Hagedorn rearing room. We have 8 3L containers of embryos and density is high!!

Fungia developmental timeseries sampling

Because we have so many Fungia larvae, I am going to do a developmental timeseries sampling at ambient. I don’t have the brain capacity to do the experiment in the incubators again–also, most of our equipment is now packed up or stored in the attic. I’m going to sample at the following time points: 1, 4, 9, 16, 20, 24, and 36 hpf. Like the Mcap experiment, 6 replicates will be taken for 3 metrics (physiology, molecular and size; 18 tubes per sampling time point). I will be collecting straight from the 3L containers, so there won’t be any 50 mL falcon tubes or cell strainers involved. The larvae are pretty concentrated so I feel confident in getting enought for each metric. The only downside is that I won’t know approximately how many are in each sample tube. For each tube, I’m collecting 100 uL of concentrated embryos.

At each time point, I will also measure the temperature and pH in each of the 8 containers of larvae.

Today, sampling took place at 20:35. I did not record the containers that I collected each sample from. Here is the environmental data:

Date	Time	BowlID	pH_mV	pH_nbs	temp_C	sal_psu	Tris_date	Notes
20230705	21:00	555	-45.5	7.95	25.99	33	20230617
20230705	21:00	629	-45.9	7.96	26	33	20230617
20230705	21:00	464	-38.1	7.82	25.92	33	20230617
20230705	21:00	879	-38.6	7.83	26.07	33	20230617
20230705	21:00	774	-47.4	7.99	26.13	33	20230617
20230705	21:00	634	-35	7.77	26.06	33	20230617
20230705	21:00	799	-40.7	7.87	26.04	33	20230617
20230705	21:00	682	-48.2	8	25.94	33	20230617

Sampling also took place at 23:37 from containers 682, 799, 464, 879, 774, and 555. Here is the environmental data:

Date	Time	BowlID	pH_mV	pH_nbs	temp_C	sal_psu	Tris_date	Notes
20230705	23:28	555	-51	8.05	25.38	33	20230617
20230705	23:28	629	-52.6	8.07	25.38	33	20230617
20230705	23:28	464	-52.9	8.08	25.4	33	20230617
20230705	23:28	879	-42.1	7.89	25.45	33	20230617
20230705	23:28	774	-50.3	8.03	25.59	33	20230617
20230705	23:28	634	-52.8	8.08	25.49	33	20230617
20230705	23:28	799	-52.9	8.08	25.45	33	20230617
20230705	23:28	682	-48.9	8.01	25.34	33	20230617

20230706

Fungia developmental timeseries sampling

Sampling took place at 4:20 from containers 464, 629, 682, 555, 634, and 879. Here is the environmental data:

Date	Time	BowlID	pH_mV	pH_nbs	temp_C	sal_psu	Tris_date	Notes
20230706	4:14	555	-48.7	8.01	24.92	33	20230617
20230706	4:14	629	-49.5	8.02	24.92	33	20230617
20230706	4:14	464	-48.2	8	24.94	33	20230617
20230706	4:14	879	-23.8	7.58	25	33	20230617
20230706	4:14	774	-45.7	7.95	25.14	33	20230617
20230706	4:14	634	-50.1	8.03	25	33	20230617
20230706	4:14	799	-48.1	8	24.95	33	20230617
20230706	4:14	682	-45.9	7.96	24.84	33	20230617

Embryos from the 7/4 and 7/5 spawn were cleaned this morning (as detailed above) around 8:30am.

Sampling took place at 11:18 from containers 464, 629, 774, 799, 682 and 634. The larvae are starting to become more buoyant, so it is more difficult to get a concentrated sample. For most tubes, I am now collecting 200 uL of moderately concentrated larvae. Here is the environmental data:

Date	Time	BowlID	pH_mV	pH_nbs	temp_C	sal_psu	Tris_date	Notes
20230706	11:08	555	-47.4	7.98	24.93	33	20230617	Water change at 8:30
20230706	11:08	629	-43.7	7.92	24.94	33	20230617	Water change at 8:30
20230706	11:08	464	-45.6	7.95	24.98	33	20230617	Water change at 8:30
20230706	11:08	879	-47	7.98	25.35	33	20230617	Water change at 8:30
20230706	11:08	774	-43.5	7.92	25.18	33	20230617	Water change at 8:30
20230706	11:08	634	-48.4	8	24.84	33	20230617	Water change at 8:30
20230706	11:08	799	-42.8	7.9	24.91	33	20230617	Water change at 8:30
20230706	11:08	682	-43.6	7.92	25.01	33	20230617	Water change at 8:30

Sampling also took place at 15:27 from containers 682, 879, 464, 555, 774 and 799. Here is the environmental data:

Date	Time	BowlID	pH_mV	pH_nbs	temp_C	sal_psu	Tris_date	Notes
20230706	15:18	555	-46	7.96	24.68	33	20230617
20230706	15:18	629	-43.4	7.91	24.59	33	20230617
20230706	15:18	464	-40.5	7.86	24.69	33	20230617
20230706	15:18	879	-41.9	7.89	24.94	33	20230617
20230706	15:18	774	-39.6	7.85	24.74	33	20230617
20230706	15:18	634	-46.7	7.97	24.55	33	20230617
20230706	15:18	799	-35.1	7.77	24.69	33	20230617
20230706	15:18	682	-40.7	7.87	24.74	33	20230617

Sampling also took place at 19:38 from containers 464, 629, 555, 799, 682 and 774. Here is the environmental data:

Date	Time	BowlID	pH_mV	pH_nbs	temp_C	sal_psu	Tris_date	Notes
20230706	19:30	555	-44.1	7.93	25.19	33	20230617
20230706	19:30	629	-40.8	7.87	25.09	33	20230617
20230706	19:30	464	-35.3	7.78	25.19	33	20230617
20230706	19:30	879	-32.5	7.73	25.36	33	20230617
20230706	19:30	774	-34.8	7.77	25.17	33	20230617
20230706	19:30	634	-44.8	7.94	25.04	33	20230617
20230706	19:30	799	-27.5	7.64	25.15	33	20230617	Lower pH relative to other bowls; may be due to higher density
20230706	19:30	682	-38.2	7.82	25.28	33	20230617

20230707

Last sampling time point! This happened at 7:44 from containers 879, 629, 634, 774, 555, and 464. Here is the environmental data:

Date	Time	BowlID	pH_mV	pH_nbs	temp_C	sal_psu	Tris_date	Notes
20230707	7:30	555	-39.3	7.84	24.29	33	20230617
20230707	7:30	629	35	7.77	24.3	33	20230617
20230707	7:30	464	-27.1	7.63	24.35	33	20230617
20230707	7:30	879	-16.2	7.45	24.52	33	20230617	Lower pH relative to other bowls; may be due to higher density
20230707	7:30	774	-25.3	7.6	24.39	33	20230617
20230707	7:30	634	-42	7.89	24.23	33	20230617
20230707	7:30	799	-9	7.32	24.35	33	20230617	Lower pH relative to other bowls; may be due to higher density
20230707	7:30	682	-30	7.68	24.34	33	20230617

Some of the containers had pretty low pH, this may be happening because certain containers look like they have higher densities than others.