Monthly Archives: November 2015

Porites astreoides study data

The following are collection and transplant details for Porites astreoides specimens collected during my November 2015 Belize trip.

Colonies labeled 1-19 brought back to CBC, sampled for DNA, photographed, dyed with alizarin red (10 mg/L for 12 h), fragmented, and transplanted in common garden. Select halves were also brought back to home site and marked with tags and subsurface markers. Samples 80-100 sampled in situ and preserved on site.
Past ID color depth habitat nearby landmark coordinates transplanted to CBC returned to home site underwater mark notes
1 brown 2 turtlegrass bed Man o War Cay 16 53.100, 88 6.459 y y pvc stick
2 brown 2.5 coral bar Cross Cay 16 58.381, 88 3.225 y n na
3 brown 4 patch reef Columbus Reef 16 57.193, 88 3.276 y n na
4 green 2 backreef/bommie Tobacco Reef 16 52.801, 88 4.042 y y pvc stick & subsurface float (orange)
100 green 2 turtlegrass bed Man o War Cay 16 53.100, 88 6.459 n na na interesting pillar-like morphology
99 green 2 backreef/bommie Tobacco Reef 16 51.464, 88 4.393 n na na
5 green 2 turtlegrass bed Blue Ground Range 16 49.278, 88 8.289 y y pvc stick & subsurface float (orange)
6 green 3 patch reef Blue Ground Range 16 49.183, 88 9.082 y n na
7 green 2 patch reef/shoal Blue Ground Range 16 47.785, 88 9.006 y y subsurface float
8 green 4 backreef/channel South Water Cay 16 48.697, 88 4.970 y n na
9 green 2 backreef Carrie Bow Cay 16 48.354, 88 4.998 y y subsurface float
98 brown 2 turtlegrass bed Blue Ground Range 16 49.278, 88 8.289 n na na
97 brown 6 patch reef Carrie Bow Cay 16 48.504, 88 5.161 n na na
96 ? 2 patch reef/shoal Blue Ground / Twin Cays 16 49.064, 88 7.748 n na na
95 ? 2 patch reef Blue Ground Range 16 48.487, 88 8.443 n na na
10 brown 2 sand/turtlegrass Wee Wee Cay 16 45.987, 88 8.631 y y subsurface float did not fasten with zspar
11 dark brown 1.5 turtlegrass/Porites bed Douglas Cay 16 42.406, 88 10.476 y y pvc stick
94 brown 4.5 cut/upper s&g forereef Carrie Bow Cay 16 47.990, 88 4.809 y n na samples put in RNAlater for gene expression
93 brown 4.5 cut/upper s&g forereef Carrie Bow Cay 16 47.990, 88 4.809 y n na samples put in RNAlater for gene expression
92 brown 4.5 cut/upper s&g forereef Carrie Bow Cay 16 47.990, 88 4.809 y n na samples put in RNAlater for gene expression
91 green 2 patch reef/shoal Blue Ground/Wee Wee Cay 16 46.828, 88 8.161 n na na
90 green 1.5 patch reef/shoal Stewart Cay/Blue Ground 16 45.917, 88 9.916 n na na
89 brown 1.5 patch reef Douglas Cay 16 42.621, 88 10.547 n na na
88 green 2 patch reef Northeast Cay 16 41.351, 88 11.422 n na na
87 brown 2 patch reef/shoal Co Cat Cay 16 39.940, 88 12.017 n na na
86 green 2 patch reef Manatee Cay 16 39.856, 88 11.289 n na na
85 green 2 patch reef Elbow Cay 16 40.892, 88 10.396 n na na
84 green 1.5 patch reef/shoal Between Douglas and Elbow Cays 16 41.776, 88 9.353 n na na
12 green 1.5 patch reef/shoal west of South Reef 16 44.896, 88 6.500 y y subsurface float
83 green 1.5 patch reef/shoal west of South Reef 16 43.988, 88 6.462 n na na
82 green 2 patch reef/shoal northwest Gladden Spit 16 41.016, 88 5.796 n na na
81 green 2 backreef northwest Gladden Spit 16 41.670, 88 4.539 n na na
80 brown 2 backreef South Cut 16 42.987, 88 4.773 n na na
13 green 2 backreef South Reef 16 45.286, 88 4.707 y y subsurface float colony badly fragmented; mulitple (4) small fragements transplanted at CBC
14 brown 1 backreef south of Curlew Cut 16 46.600, 88 4.744 y y subsurface float
15 brown 2 backreef Carrie Bow Cay 16 48.126, 88 4.968 y n na
16 green 0.75 backreef – near exp plot Carrie Bow Cay 16 48.167, 88 4.857 y y na colony collected adjacent to commong garden, treated same as all others, and re-planted in common garden
17 brown 0.75 backreef – near exp plot Carrie Bow Cay 16 48.167, 88 4.857 y y na colony collected adjacent to commong garden, treated same as all others, and re-planted in common garden
18 green 0.75 backreef – near exp plot Carrie Bow Cay 16 48.167, 88 4.857 y y na colony collected adjacent to commong garden, treated same as all others, and re-planted in common garden
19 green 0.75 backreef – near exp plot Carrie Bow Cay 16 48.167, 88 4.857 y y na colony collected adjacent to commong garden, treated same as all others, and re-planted in common garden
Map2

DNA methylation inhibition experiment

I arrived at Carrie Bow Cay late in the day on 11/11 and quickly got to work setting up an experiment attempting inhibition of DNA methylation in corals using 5-aza-2′-deoxycytidine, also known as Decitabine. Once incorporated into DNA, this cytosine analog inhibits de novo methylation. The goal is to identify a causative role of DNA methylation in coral thermal tolerance plasticity. If corals treated with Decitabine do not achieve the same level of thermal tolerance plasticity as untreated corals, this would indicate that DNA methylation may play a role in thermal tolerance plasticity (or buffering, as may be the proper term).

For the experiment, I chose the branching coral Porites porites because branch tips (“nubbins”) can be easily and cleanly cut from colonies and glued to a substrate of choice. I cut four replicate nubbins from four different colonies in the backreef (1-2 m depth) at Carrie Bow Cay. I glued the nubbins to small petri dish lids with A-788 compound, then buoyant weighed and photographed them. This morning, I placed the nubbins in their respective treatments. There are two water baths, one filled with flow-through ambient seawater, and the other with a much smaller amount of flow through water, plus a heater and a water pump for mixing. Thus the experiment is a factorial arrangement with treatments ambient (A), heated (H), Decitabine (D), and control (C).

A stock solution of Decitabine (FW = 228.21) at a concentration of 219 uM was made by dissolving 10 mg of the compound in 200 ml filtered seawater. This stock solution is kept refrigerated and I anticipate making a new stock approximately every other day. For a final concentration of 10 uM in the experimental beakers (based on the concentration used by Amarasinghe et al. 2014 in bumble bee apiary syrup), I added 3.6 ml of the stock into beakers filled with 80 ml seawater. I will do water changes twice daily.

I placed the corals in the experimental beakers today at 9:30 AM, and added Decitabine at around 10:30. The beakers were covered with a glass plate to reduce evaporation and rain water entry. This will also attenuate some light which is fine since the experiment is outdoors.

Here is a photo of the setup, with ambient on the left and heated on the right, plus a closeup of a beaker with coral nubbin.

IMG_8044IMG_8046

Temperature record

Date time ambient heated heater setting °F heater setting °C
13-Nov 10:30 26.4 27.6 81 27.22
13-Nov 12:00 26 27 81 27.22
13-Nov 15:15 26.5 27.4 81 27.22
13-Nov 17:00 26.5 27 81 27.22
13-Nov 20:00 26.1 27.8 81 27.22
13-Nov 22:00 25.9 27.9 81 27.22
14-Nov 9:00 26.3 27.3 81 27.22
14-Nov 11:45 26.7 27.7 81.5 27.50
14-Nov 14:30 26.3 27.3 81.5 27.50
14-Nov 17:30 26.3 27.5 81.5 27.50
14-Nov 20:00 25.3 27.4 81.5 27.50
15-Nov 8:30 26.3 28.3 81.5 27.50
15-Nov 11:00 26.3 28.4 81.5 27.50
15-Nov 13:00 nd 28 82 27.78
15-Nov 19:00 25.6 27.8 82 27.78
16-Nov 8:30 26.9 28 82 27.78
16-Nov 10:00 27.2 28.7 83 28.33
16-Nov 12:00 26 27.2 83 28.33
16-Nov 13:00 26.5 29 83 28.33
16-Nov 18:00 26.9 27.3 83 28.33
16-Nov 20:30 26.7 28.3 83 28.33
16-Nov 21:45 25.8 27.9 84 28.89
17-Nov 7:45 26.4 28 84 28.89
17-Nov 10:30 27.4 29 84.5 29.17
17-Nov 12:00 27.5 29.8 84.5 29.17
17-Nov 18:00 27.1 27.8 84.5 29.17
18-Nov 7:00 26.5 29 84.5 29.17
18-Nov 13:15 27.8 30.6 84.5 29.17
18-Nov 19:00 27 29 84.5 29.17
18-Nov 22:45 27.6 28.2 84.5 29.17
19-Nov 6:30 27.5 29.4 84.5 29.17
19-Nov 10:30 28 30 85.5 29.72
19-Nov 11:30 27.6 30.3 85.5 29.72
19-Nov 19:15 26.5 28 85.5 29.72
19-Nov 22:00 26.9 30.4 85.5 29.72
20-Nov 9:00 27.4 30.5 85.5 29.72
20-Nov 12:00 27.3 30 85.5 29.72
20-Nov 7:30 26.4 29.5 85.5 29.72
21-Nov 6:45 na 31 89.5 31.94
21-Nov 15:00 na 31.4 89.5 31.94
21-Nov 20:15 na 31.2 89.5 31.94
22-Nov 7:00 na 30.5 89.5 31.94
22-Nov 10:15 na 31 90 32.22
22-Nov 19:00 na 31.9 90 32.22
23-Nov 7:00 na 32.3 90 32.22
23-Nov 13:00 na 31.5 90 32.22
23-Nov 17:30 na 31 90 32.22
24-Nov 8:30 na 30.4 91 32.78
24-Nov 12:00 na 31 91 32.78
24-Nov 16:00 na 31 91 32.78

Temp

Buoyant weights of P. porites nubbins
ambient (A), heated (H), Decitabine (D), and control (C)
Colony Water Treatment Initial weight (g) Final weight (g) final-initial/final *100
1 A C 3.9 3.94 1.025641026
1 A D 3.45 3.47 0.579710145
1 H C 4.48 4.52 0.892857143
1 H D 2.93 2.96 1.023890785
2 A C 1.64 1.67 1.829268293
2 A D 2.63 2.73 3.802281369
2 H C 1.4 1.43 2.142857143
2 H D 2.57 2.62 1.945525292
3 A C 2.64 2.67 1.136363636
3 A D 2.27 2.3 1.321585903
3 H C 3.56 3.64 2.247191011
3 H D 1.79 1.8 0.558659218
4 A C 1.72 1.75 1.744186047
4 A D 2.82 2.84 0.709219858
4 H C 2.91 2.92 0.343642612
4 H D 2.13 2.15 0.938967136

 

weights