Dataset: Disease Exposure Experiment
Deployment: MOTE_Muller_SummerlandKey-Summer2015

Sampling and Analytical Methodology:
Three trials were conducted to test the disease susceptibility of 15 different genotypes of Acropora cervicornis in August 2015. Approximately 10 replicates of each genotype were collected from the Mote offshore coral nursery. Replicates consisted of approximately 5cm branch tips collected from different individuals of the same genotype. Fragments were attached to glass microscope slides using super glue and allowed to acclimate in outdoor raceways for 2 days prior to treatment. One replicate of each genotype was placed within a single five gallon glass tank, holding approximately 10 liters of water. A total of ten tanks were used, five for the disease homogenate applications, and five for healthy homogenate applications. Temperature within the raceways were kept at ~28.5C; pH (8.0), and salinity (34ppt) were measured daily and remained constant throughout the trials.

After the acclimation period, fragments of diseased corals were collected from a natural reef located at 24'31.640N, 81'29.938W (Permit: FKNMS 2015-84). Fragments showing active signs of tissue loss were snipped off of the donor colony, placed in plastic containers and brought back to the boat for transport to Mote TRL. Upon return to the lab, the diseased corals were airbrushed with filtered sterilized seawater and collected in 50 ml falcon tubes. Tissue adjacent to the disease edge, up to 4 cm away was removed through this method and collected to create a disease homogenate. Healthy homogenates were created from nursery coral fragments. Approximately 10, 5 cm long fragments were airbrushed and collected for the healthy homogenate applications for each trial. Each tank, holding 10 L of water, were treated with either 100 ml of disease homogenate or 100 ml of healthy homogenate.

For the next 8 days the presence of tissue loss was identified from each individual replicate. Photographs of each coral were taken daily. The rate of tissue loss was calculated (cm/day) from each replicate using ImageJ software.

An imaging pulse amplitude fluorometer was used after the 2 day acclimation period to quantify the photochemical efficiency of each replicate coral. The photochemical yield was quantified and a light reaction curve was applied.

Snips, approximately 2 cm in size, were taken from two random replicates within each genotype for Symbiodinium sequencing. Preliminary analyses indicate that all genotypes contain only A3 clade symbiodinium, but they may harbor different genotypes of the symbiont. These samples were flash frozed at -80C and stored until processing. DNA was isolated and extracted from 2 - 3 polyps worth of tissue using the Qiagen DNAeasy DNA isolation kit. Samples will be sent to Dr. Iliana Baums at Penn State University for microsatellite analysis and identification of possible variations in symbiont genotypes within coral genets.

In September of 2015, when corals were visibly bleached from anomalous high water temperatures, a fourth trial was conducted using the same methods described above to test the difference in susceptibility during a thermal stress event. Diseased fragments were collected from the same location as the original trial.

Changes from original proposal: Ideally, all 48 genotypes would have been tested during the summer of 2015. However, even in August 2015 (prior to peak bleaching), several coral genotypes were suffering from thermal stress already. Only the 15 genotypes collected and used for experimentation did not visually appear to be initally stressed during August. Collaborator and nursery manager, Erich Bartels, did not encourage the collection of the other genotypes as they were already thermally stressed; however, Erich agreed to provide the same 15 genotypes in September 2015 even though they were visibly bleached for the sake of a novel research experiment.