During the September/October 2005 cruise, samples were collected from multiple reef sites around Necker Island, French Frigate Shoals, Maro Reef, Pearl and Hermes Atoll, Midway Atoll, and Kure Atoll. During the May/June 2006 cruise, samples were collected from Kaneohe Bay (Oahu), Nihoa, French Frigate Shoals, Gardner Pinnacles, Johnston Atoll, and American Samoa.

Coral colonies judged as non-diseased by visual inspection were sampled. A stainless steel chisel was used to remove three dime-sized pieces of coral from each colony that consisted of coral tissue, overlying mucous layer, and underlying skeleton. The three sub-samples from each colony were put into sterile bags (Whirl-pak; Nasco, Fort Atkinson, WI), placed on ice, and transported back to the ship where they were frozen. Temperature and depth below sea surface were recorded at each site with a dive computer.

Seawater samples were also collected to characterize planktonic bacterial communities in the surrounding environment. One liter of seawater was collected adjacent to coral heads, placed on ice, and filtered on board the research vessel through a series of 25 mm diameter, 1.6 μm nominal pore-sized GF/A glass microfiber filters (Whatman International Ltd., Piscataway, NJ) and 13 mm diameter, 0.2 μm pore-sized polyethersulfone membrane filters (Supor-200; Pall Corp., East Hills, NY). Filters were then stored frozen in 250 μL of DNA lysis buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 1.2% v/v Triton X100) (Suzuki et al. 2001). All samples were transported to the Hawaii Institute of Marine Biology where they were further processed for DNA analysis.

DNA extraction and T-RFLP of bacterial SSU rRNA genes

Coral samples were thawed on ice and a flame-sterilized core borer was used to remove a 6-mm diameter, 6-mm deep core from each of the three subsamples per colony. Cores from each colony were placed into a sterile bag containing 2 mL of 0.2 μm-filtered 10X Tris EDTA (100 mM Tris, 10 mM EDTA) buffer solution (pH 7.4). An air gun outfitted with a sterile pipette tip was used to remove coral tissue from the skeleton. The coral tissue slurry was centrifuged at 19,900 RCF for 30 min at 4˚C. The supernatant was removed and the remaining sample pellet was frozen at -80˚C until further processed.

Genomic DNA was extracted from coral and seawater samples using the PowerSoil DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA) according to the manufacturer’s protocol. Total genomic DNA yield was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen Corp., Carlsbad, CA, USA) and SpectraMax M2 plate reader (Molecular Devices Corp., Sunnyvale, CA, USA).

Approximately 69 ±34 ng of genomic DNA was used as template for polymerase chain reaction (PCR) amplification of bacterial small subunit (SSU) ribosomal RNA (16S rRNA) genes in preparation for terminal-restriction fragment length polymorphism (T-RFLP) analysis (Liu et al. 1997).

The 50 µl PCR reactions included 1X MasterTaq reaction buffer (Eppendorf AG, Hamburg, Germany), 2.25 mM Mg2+, 0.5X TaqMaster reaction enhancer (Eppendorf), 0.2 mM each of the fluorescently labeled general bacterial SSU rRNA gene oligonucleotide primer 27F-B-FAM (5’-FAM-AGRGTTYGATYMTGGCTCAG-3’) and universal SSU rRNA gene oligonucleotide primer 1492R (5’-GGYTACCTTGTTACGACTT-3’; Lane, 1991), 0.2 mM of each deoxynucleotide (Promega, Madison, WI), 2.5 units of MasterTaq DNA polymerase (Eppendorf), and sterile water. PCR reactions were carried out in a MyCycler thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) with an initial incubation of 3 min at 95°C, followed by 30 cycles of 30 sec at 95°C, 1 min at 65°C (decreasing by 0.5°C per cycle), and 2 min at 72°C. Reactions were concluded with 10 cycles of 30 sec at 95°C, 1 min at 50°C, and 2 min at 72°C, and 1 cycle of 30 sec at 95°C, 1 min at 50°C, and 20 min at 72°C. Template-free PCR reactions were used as negative controls.

Fluorescently labeled PCR amplicons were purified using the QIAquick Multiwell PCR Purification System (Qiagen Inc., Valencia, CA) and approximately 100 ng of each purified amplicon was digested in a 10 µL reaction containing 5 units of HaeIII restriction endonuclease (Promega, Madison, WI). After a 6-h incubation at 37ºC, digests were purified via gel filtration chromatography using the Millipore MultiScreen Assay System (Millipore Corp., Billerica, MA) paired with Sephadex G-50 Superfine (GE Healthcare, Piscataway, NJ). Purified products were adjusted to a final concentration of 30 ng µL-1 and separated on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).