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.
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 |
| 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 |
