Sample Collection:
Following the disease outbreak, fifteen samples of Pseudodiploria strigosa corals were collected in the Flower Garden Banks National Marine Sanctuary at the West Flower Garden Bank (Buoy 2: 27.875433, -93.813933 and Buoy 5: 27.875317, -93.814817) and East Flower Garden Bank (Buoy 2: 27.9086, -93.597183). Samples were collected on SCUBA using a hammer and chisel at a depth range of 61 to 70 feet. Corals (n = 10 colonies) were sampled in situ as sets of diseased and apparently healthy P. strigosa, where two samples were collected per diseased coral (DD: adjacent to the tissue loss lesion, n = 5; HD: apparently healthy tissue on the same coral, n = 5) and one sample was collected from a neighboring apparently healthy conspecific coral (HH; n = 5). Each sample was divided into three fragments that were preserved for histopathology (data not included herein), transmission electron microscopy (TEM), and DNA/RNA sequencing. Histopathology tissue samples were fixed in zinc-buffered formalin (Z-fix concentrate from Anatech Ltd. diluted with filtered seawater) for 24 hours, washed with seawater for 24 hours, then stored in 70% ethanol for transport to Louisiana State University, Baton Rouge, LA, USA. TEM samples were preserved in 750 microliters (µL) of 2% PFA in 3X PBS at 4 degrees Celsius (ºC) for transport to Rice University, Houston, TX, USA. For DNA/RNA extraction, samples were collected, flash frozen in liquid nitrogen, and stored at -150ºC for transport to the University of Texas at Arlington, Arlington, TX, USA.
Transmission Electron Microscopy (TEM) Imaging:
Coral fragments ~3 square millimeters (mm²) in size (fixed in 750 µL of 2% PFA in 3X PBS) were decalcified in 1000 µL of 1.5% ascorbic acid for 7-14 days, replacing the ascorbic acid solution daily until the entire skeleton was dissolved. The decalcified samples were fixed in Karnovsky's fixative for at least 24 hours before being post-fixed and processed according to (Howe-Kerr et al., 2023). Electron micrographs were imaged using a 120 kV JEOL JEM-1400Flash Transmission Electron Microscope equipped with a high-contrast pole piece and a 15 megapixel AMT NanoSprint15 sCMOS camera. Electron micrographs showed 438 total Symbiodiniaceae cells: 123 from HH tissue (20-30 cells per sample), 142 from HD tissue (23-38 cells per sample), and 173 from DD tissue (27-46 cells per sample). Electron micrographs were labelled by cell ID and grouped into separate pdf files per sample.
Nucleic Acid Extraction and Sequencing:
Subsets of the fifteen P. strigosa samples were first extracted for total RNA using a pea-sized amount of coral tissue harvested from the frozen samples using stainless steel sterilized bone cutters. For DD samples, the tissue was taken ~1 to 2 centimeters from the visible lesion line. Extractions were conducted using the Invitrogen RNAqueous Kit (AM1914). Potential DNA and chromatin contamination was removed from each sample using the Ambion DNase I (RNase-free) kit from Invitrogen (Life Technologies AM2222). Total RNA aliquots were sent to Novogene Co., LTD for library preparation and paired-end sequencing. All 15 P. strigosa samples had an RNA integrity number (RIN) ≥ 4 and were pre-processed with poly-A tail enrichment to capture all eukaryotic reads. Library prep was completed using the NEBNext Ultra II RNA Library Prep kit by Illumina and samples were processed for 150bp, paired-end sequencing on the Illumina Novaseq 6000 platform with raw read file sizes at a minimum of 5 million.
Upon confirmation of successful RNA extraction, DNA was extracted from the remainder of each coral. Coral samples were airbrushed with a PBS solution to remove coral tissue from the skeleton. The resulting tissue slurry was spun in a centrifuge at maximum speed at 4ºC for 40 minutes or until there was a well-formed pellet with near-transparent supernatant. DNA was extracted from ~0.040 grams (g) of the pelleted coral holobiont biomass with the Qiagen DNeasy PowerBioFilm Kit (Qiagen, Inc., Germantown, MD, 24000-50) according to the manufacturer's protocol and stored at -80ºC until further processing. One 50 µL aliquot of extracted DNA was transferred to Rice University and another was transferred to Woods Hole Oceanographic Institution for sequencing of Symbiodiniaceae ITS-2 rDNA and 16S rRNA genes for bacteria/archaea, respectively.
To characterize Symbiodiniaceae composition, the ITS-2 region of Symbiodiniaceae rDNA was PCR amplified with the Sym_VAR primer set (Sym_VAR_5.8SII and Sym_VAR_REV; Hume et al., 2018). Each 16 µL PCR reaction contained: 1 µL DNA template, 9 µL EconoTaq PLUS GREEN 2X Master Mix (Biosearch Technologies), 0.42 µL each of forward and reverse primer at 100 µM, and 5.16 µL DNAse/RNAse-free water. PCR reactions were cycled as: initial denaturation at 94°C for 3 minutes; followed by 35 cycles of 94°C for 75 seconds, 58°C for 75 seconds, and 74°C for 90 seconds; and a final extension at 74°C for 7 minutes, before holding at 4°C. Library preparation and ITS-2 sequencing were performed at Oregon State University's Center for Quantitative Life Sciences (OSU CQLS, Corvallis, OR, USA) with 2x300 bp paired-end reads on an Illumina MiSeq platform. Blanks (nuclease-free PCR-grade water) were used as negative controls during sequencing.
To characterize bacteria and archaea, single barcoded PCR reactions were performed to amplify the V4 region of the small subunit rRNA gene using primers 515FY and 806RB (Apprill et al., 2015; Parada et al., 2015). As a sequencing control, we used genomic DNA from Microbial Mock Community B (even, low concentration), v5.1L, HM-782D, and nuclease-free PCR-grade water for the negative control. Each 50 μL PCR reaction contained: 2 μL DNA template, 0.5 μL GoTaq DNA Polymerase (Promega), 1 μL of each forward and reverse primer at 10 micromolar (μM), 1 μL of 10 millimolar (mM) deoxynucleoside triphosphate (dNTP) mix (Promega), 5 μL MgCl₂, 10 μL GoTaq 5X colourless flexi buffer (Promega), and 29.5 μL nuclease-free water. The PCR cycling conditions were as follows: initial denaturation at 95°C for 2 minutes; followed by 34 or 37 cycles of 95°C for 20 seconds, 55°C for 15 seconds, and 72°C for 5 minutes; and a final extension at 72°C for 10 minutes, before holding at 4°C. We used 34 or 37 cycles for samples to obtain comparable amounts of amplified DNA for subsequent purification and sequencing. Barcoded PCR products were purified using the MinElute PCR Purification Kit (Qiagen, Inc., Germantown, MD), and concentrations were measured using a Qubit 2.0 fluorometer. Purified samples were diluted to 1 nanogram per microliter (ng/μL) and pooled for sequencing. Sequencing was performed with 2x250 bp paired-end reads on an Illumina MiSeq platform at the University of Illinois at Urbana-Champaign (Kozich et al., 2013). During analysis of the resulting fastq files, two samples (HD and DD tissues from coral 007) were excluded from analysis due to a mislabeling error during laboratory processing.
