In the summer of 2022, 20 ramets each of the Acropora cervicornis genotypes ML-AC-36 and ML-AC-46 were collected from Mote’s in situ coral nursery and subjected to three weeks of elevated nitrate, ammonium, and phosphate in the form of a slow-release fertilizer. The duration and concentration of this exposure were selected based on prior experiments conducted at Mote Marine Laboratory, which were found to alter microbial community profiles and growth rates in A. cervicornis (Klinges et al., 2022, 2023). During experiment, corals were held in 2-gallon aquaria with 5 corals per aquarium. As Mote Marine Laboratory’s experimental aquarium system is plumbed into nearshore coastal water and thus has a higher nutrient load than the reef, an additional subset of 18 ramets of each genotype was collected from Mote’s in situ coral nursery immediately prior to disease challenge to evaluate the impact of nearshore water on disease response. All ramets in the disease group were subjected to disease challenge in the form of a tissue homogenate produced from diseased fragments of random genotypes of A. cervicornis. Ramets in a comparative unexposed group were exposed to a homogenate produced from healthy fragments of random genotypes of A. cervicornis. To evaluate the effect of Aquarickettsia infection; nutrient enrichment; disease exposure; and the combination of these factors on coral immune function, a number of immune related proteins and antioxidants were measured using microplate assays from a total of 55 samples taken prior to disease exposure and following disease exposure as corals either resisted or developed disease. Total host protein and the antioxidant superoxide dismutase were found to be higher in healthy corals regardless of genotype or nutrient enrichment. Phenoloxidase and prophenoloxidase were higher in concentration in diseased samples compared to healthy samples. Disease-exposed but apparently healthy corals had higher superoxide dismutase, prophenoloxidase, and peroxidase activity than either healthy unexposed corals or diseased corals despite total protein concentrations that were lower than healthy unexposed corals.

Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • Problem Description
• Data Files
• Supplemental Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

Proteins were extracted over ice using a Paansche airbrush with coral extraction buffer (50 mmol tris buffer, pH 7.8, with 0.05 mmol dithiothreitol). Tissues were then homogenized using a VWR 200 tissue homogenizer with a medium saw tooth generator for 60 seconds on ice. Samples were then left on ice for 10 minutes. From the resulting extract, 1 mL was reserved for melanin analysis. The remaining volume was centrifuged for 5 minutes at 4°C and 3500 RPM in an Eppendorf centrifuge 5810R. The resulting supernatant, or coral extract, was split into two ~2 mL aliquots mL which were frozen and stored at -80°C. 

Total host protein (total HP) in each sample was determined using Bradford reagent standardized to BSA. These concentrations were used to standardize all biochemical assays conducted on the samples. 

All colorimetric assays were run in triplicate on 96-well plates using a Synergy H1 Hybrid Multi-Mode microplate reader and Gen5 software. Samples were stored at -80°C and thawed immediately prior to processing.

To measure peroxidase (POX), 20 μL of sample were diluted with 20 μL 10 mM phosphate buffer, pH 6.0. Then 25 μL of 25 mM guaiacol in 10 mM phosphate buffer, pH 6.0, was added to each well. The reaction was initiated with 20 μL of 20 mM hydrogen peroxide and optical density was measured every 34 seconds for 15 minutes at 470 nm. Results were calculated as change in absorbance per minute, normalized according to mg of protein.
To measure prophenoloxidase (PPO), 20 μL of sample were diluted with 20 μL 50 mM phosphate buffer, pH 7.0. Next, samples were incubated for 30 minutes in 25 μL of trypsin (0.1 mg/mL). Just prior to the assay, 30 μL of 10 mM L-1,3- dihydroxphenylalanine (L-dopa) was added to each sample. Absorbance was then read every minute for 20 minutes at 490 nm at 26C. Results were calculated as change in absorbance per minute at the steepest point of the curve, normalized according to mg of protein.
To measure phenoloxidase (PO), 20 μL of sample were diluted with 20 μL 50 mM phosphate buffer, pH 7.0. Next, samples were incubated for 30 minutes in 25 μL of molecular grade water. Just prior to the assay, 30 μL of 10 mM L-1,3- dihydroxphenylalanine (L-dopa) was added to each sample. Absorbance was then read every minute for 20 minutes at 490 nm at 26°C. Results were calculated as change in absorbance per minute at the steepest point of the curve, normalized according to mg of protein.

A Superoxide Dismutase (SOD) Activity Assay Kit from Sigma Aldrich was used to measure superoxide dismutase activity following manufacturer's protocols. Briefly, 20 μL of coral extract was incubated with WST dye and 20 μL xanthine oxidase for 30 minutes at 25°C. A standard curve was performed using the provided SOD enzyme. Percent inhibition of absorbance was then measured at 450 nm by comparing the absorbance of the samples to that of the control wells. Activity is reported as superoxide dismutase activity standardized by mg protein.

Peroxidase: Results were calculated as change in absorbance per minute at the steepest point of the curve, normalized according to mg of protein.

Prophenoloxidase: Results were calculated as change in absorbance per minute at the steepest point of the curve, normalized according to mg of protein.

Phenoloxidase: Results were calculated as change in absorbance per minute at the steepest point of the curve, normalized according to mg of protein.

Superoxide dismutase: Percent inhibition of absorbance was then measured at 450 nm by comparing the absorbance of the samples to that of the control wells. Activity is reported as superoxide dismutase activity standardized by mg protein.

Coefficient of variation and standard deviation of mean were used to evaluate whether samples performed in triplicate should be rerun. A coefficient of variation less than 30 was considered to be acceptable. In triplicate measures with an obvious outlier, samples were rerun in triplicate and averages were made across both runs, omitting the outlier.

Coverage: at Oregon State University and in the Florida Keys at Mote Marine Laboratory

NSF Award Abstract:
Historically one of the most abundant reef-building corals in Florida and the wider Caribbean, the staghorn coral, Acropora cervicornis, is now listed as critically endangered primarily because of previous and reoccurring disease events. Understanding the holistic mechanisms of disease susceptibility in this coral is a top concern of practitioners engaged in conservation and restoration. The investigators recently discovered a group of parasitic bacteria common within the microbial community of A. cervicornis that can reduce the growth and health of corals when reefs are exposed to nutrient polluted waters. Determining how interactions among the coral host, this parasitic microbe, and the environment are linked to disease susceptibility provides critical insight and greater success of future restoration efforts. Yet the complexity of animal microbiomes and the contextual nature of disease make it difficult to identify the specific cause of many disease outbreaks. In this project, the investigators conduct experiments to explore the interactions among different genetic strains of coral and these bacteria in various nutrient scenarios to better understand how this bacterium affects the susceptibility of staghorn coral to diseases. This project also characterizes the genomics, host range, and local and global distribution of this bacterial coral parasite to determine how its evolutionary history and physiology drive disease susceptibility in this important coral species. The project trains two postdocs, one technician, and seven students (one graduate, six undergraduates) in integrative sciences that span marine science, physiology, genetics, microbiology, omics, and statistical modeling. A research-based after school program in Florida is expanded to include microbiology and create a new program module called Microbial warriors, with a focus on women in science. The investigators produce documentary style films and outreach materials to broadly communicate the project science and conservation efforts to local and national communities via presentations at Mote Marine Lab and the Oregon Museum of Science and Industry. This project is co-funded by the Biological Oceanography Program in the Division of Ocean Sciences and the Symbiosis, Defense, and Self-recognition Program in the Division of Integrative Organismal Systems.

The investigators recently identified a marine Rickettsiales bacterium that, in corals, can be stimulated to grow in the presence of elevated nitrogen and phosphorous species. Based on genomic reconstruction and phylogeography, this bacteria is classified as a novel bacterial genus, Candidatus Aquarickettsia, and showed that it is broadly associated with scleractinian corals worldwide. Importantly, using a model system, the endangered Acropora cervicornis coral, the team has also shown that the growth of this bacterium in vivo is associated with reduced host growth and increased disease susceptibility. This project aims to more completely evaluate the mechanisms behind and impacts of these inducible infections on coral physiology and host-bacterial symbiosis. The investigators conduct nutrient dosing experiments on different coral genotypes with various Rickettsiales abundances. Using a range of omics and microscopy techniques, the team quantifies the resulting effects on holobiont phenotypes. The investigators are also comparing the genomes of these bacteria in the different Acroporid hosts and other coral genera to evaluate facets of the bacterium's evolutionary history, as well as to identify possible mechanisms of its proliferation, virulence, and host specificity. This interdisciplinary project mechanistically links nutrients to temporal changes in host, algal symbiont, and bacterial parasite physiology and also explain why there is natural variation in these responses by exploring how host and parasite genotypes and growth dynamics combined with environmental contextuality alter holobiont phenotypes.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.