19 March 2022

Heatwave Experiment Sampling

Danielle, Lauren, and Alex went out early in the morning to our Mahana site to collect for the heatwave experiment. 12 total distinct genotypes of Acropora pulchra were sampled and split in half for an ambient and a hot treatment with each half being ~10-15 inches wide to ensure the colonies could be reproductively viable for spawning in October 2022. Each colony was tagged with the genotype number and either a red tag or a blue tag to represent ambient and hot treatment. While Danielle and Alex were collecting, Lauren took bleaching score photos of each individual colony. From the same genotype (but separate from the colonies), 14 small ~ 3-4 inch fragments were collected for 7 to go into the hot treatment and 7 to go into the ambient to be sampled over 5 distinct timepoints during the heatwave experiment (initial, after acclimation, after ramp, after heatwave, and after decline), with one extra for mortality, and one extra to go onto the CRIOBE coral trees for future experiments. Colonies and bags were transported in fresh seawater in covered coolers before being taken back to Gump.

Bleaching Score Photos - Colonies

All bleaching score photos were downloaded off of the Putnam Lab Canon G7X camera number 2 and uploaded to the Heatwave_Bleaching_Score google drive for the initial heatwave observations.

Respirometry - Photosynthesis and Respiration

To measure photosynthesis and respiration for our initial timepoint 1 fragments (n=24) we followed the Putnam Lab respirometer manual. Since our set up in Mo’orea has two six chamber respirometer stands, we did three separate runs with n=8 fragments with one blank on each stand for a total of n=10 chambers per run. We first took molecular samples of each fragment with one ~0.5-1 cm biopsy preserved in 1 mL of RNA/DNA shield and one ~0.5-1 cm biopsy snap frozen in liquid nitrogen and stored in the Gump Molecular -40 C freezer. We set our light levels to ~610 μmol photons m−2 s−1 (51% on the AI lights) following our PI cuvre calculations to measure photosynthesis for ~20 minutes or until a consistent reading was achieved and then we turn off the lights and conducted light-enhanced dark respiration for ~20 minutes or until a consistent reading was achieved. After each trial was completed, volumes were tamen from the chambers and the physiological fragments were snap frozen and stored in the Gump Molecular -40 C freezer in whirl paks.

Data

All respirometry data was uploaded to the timepoint 1 heatwave google drive folder and the Gametogenesis GitHub Repo.