Table of Contents

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

Sequence data can be found in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under accession number SRP267474 with the associated BioProject PRJNA639601. Additional metadata related to the NCBI submission are provided in Supplemental File "MIMARKS.specimen.host-associated.5.0.xlsx".

A total of two replicate fragments (ramets) from 16 genotypically distinct host colonies (genets) were collected from the Mote Marine Laboratory in situ coral nursery in August 2015. In September 2015, another set of two ramets from 15 of the same genets of Acropora cervicornis were collected from the Mote Marine Laboratory in situ coral nursery (one genotype, G20, was not available due to high mortality rates). By this time the nursery corals had been experiencing anomalously high water temperatures reaching approximately ~2° Celsius (C) above historical averages, represented by 8 degree heating weeks (DHWs) under NOAA's Coral Reef Watch products (http://www.coralreefwatch.noaa.gov). These ramets were the same individuals utilized in Muller et al. (2018) to assess changes in disease susceptibility prior to and after bleaching. Genotypes were delineated in that study via microsatellite genotyping (Baums et al., 2005). When corals appeared healthy (i.e., no visual signs of bleaching) genotype 3 was completely resistant to white band disease, and genotype 7 had only 1 out of 5 replicates succumb to disease after exposure. Genotypes 3 and 7 both were resistant to white band disease even after bleaching. All ramets hosted Symbiodinium fitti, the main species of Symbiodiniaceae found in the Mote A. cervicornis nursery corals (Muller et al. 2018, Parkinson et al., 2018). All corals sampled in September were visibly bleached and showed a significant reduction in photochemical efficiency using Pulse Amplitude Modulator fluorometry (see Muller et al., 2018). Upon returning to the lab, all samples were flash-frozen in liquid nitrogen and stored in at -80°C until processing. A total of two polyps were scraped from each replicate using a sterile razor blade, and total DNA was extracted from polyp tissue using the MoBio Powersoil kit.

The bacterial community dynamics of each sample was analyzed using 16S rRNA Illumina sequencing on the MiSeq platform. Amplification of the 16S rRNA gene was conducted using the 515F-806R primer set, which targets the V4 region of the 16S rRNA, with barcodes on the forward primer (Apprill et al., 2015). A polymerase chain reaction (PCR) was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, followed by a final elongation step at 72°C for 5 minutes. PCR products were checked on a 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Angencourt Ampure XP beads (Beckman Coutler, CA, USA). Then the pooled DNA library was generated using the Illumina TruSeq DNA library preparation protocol. A PCR negative control was included in library preparation but did not produce a viable library. Paired-end sequencing was performed at MR DNA (http://www.mrdnalab.com, Shallowater, TX, USA) using a single flow cell on a MiSeq following the manufacturer’s guidelines.

Known problems/issues:
Genotype 20 was not sampled after bleaching due to mortality.

Demultiplexing and barcode removal was performed using sabre (v1.0) (Copyright © Nikhil Joshi, UC Davis), during which a total of 7,499,952 reads with no barcode match were discarded from the initial pool of 15,577,446 reads. A total of 8,077,494 reads across 62 samples were subsequently processed using DADA2 (v1.16) (Callahan et al., 2016) in R (v1.1.383) (R Development Core Team, 2008). DADA2 identifies unique 16S rRNA sequences at single-nucleotide resolution (amplicon sequence variants, ASVs) using a core denoising algorithm that models substitution errors in Illumina reads. Based on quality plots, forward and reverse reads were truncated at their 3′ end at 260 and 210 base pairs, respectively. Sequences were truncated at the first position having a quality score less than or equal to 10, and reads with a total expected error of >1 or with the presence of Ns were discarded, resulting in a total of 6,178,780 reads. Amplicon sequence variants (ASVs) were inferred from unique reads and paired-end reads were subsequently merged to equal 3,089,390 reads. ASVs that did not match a target length of 292 ± 2 (77 ASVs) were discarded. Two-parent chimeras (bimeras) were removed and taxonomy was assigned at 100% sequence identity using the Silva reference database (v132) in order to preserve the high resolution of ASV data (Quast et al., 2012). The Silva taxonomic classifications for the genera MD3-55 and HIMB11 were changed to Ca. Aquarickettsia and Roseobacter, respectively, in accordance with current literature identifications (Klinges et al., 2019, Durham et al., 2014). The resulting ASV table contained 2,979 unique ASVs across 62 samples and was imported into phyloseq (v1.30.0) (McMurdie & Holmes, 2013). A total of 102 ASVs were then removed from the dataset that were annotated as mitochondrial or chloroplast sequences, corresponding to 613 and 25,100 reads, respectively. Although there were no singletons in the dataset, 2,052 unique ASVs were present in only one sample each. Next, ASVs with a total count across the dataset in the bottom first-quartile (count = 29) were removed resulting in a total of 2,618,743 total reads, 2,133 unique ASVs, and a median sample depth of 36,970 reads.

- Imported original file named "BCO-DMO Data_acropora_cervicornis_bleaching.xlsx" into the BCO-DMO system.
- Added a column for "collection_date" in YYYY-MM format.
- Saved the final file as "847425_v1_bleaching_sequences.csv".

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.