Dataset: coral microbe T-RF - temp stress

Sample Collection and Experimental Design: On August 4, 2007, approximately 45 branch tips of Porites compressa (Dana 1846) 3 to 5 cm in length and 1 to 1.5 cm in diameter were removed from each of three coral colonies judged as nondiseased by gross visual assessment in central Kaneohe Bay off of the island of Oahu, Hawaii, in the Pacific Ocean. Source colonies were sampled at a depth of approximately 4m and were located within 10m of each other. Three fragments from each colony were immediately frozen in liquid nitrogen for analysis of bacterial community structure associated with the source colonies. The remaining fragments were immediately placed in containers with aerated seawater and transported back to the Hawaii Institute of Marine Biology (HIMB). In addition to the coral sampling, water samples were collected to characterize the bacterial communities in the planktonic environment surrounding the corals. Approximately 1 L of seawater was collected ~1.5m above the reef, filtered through 25mm diameter, 0.2 µm pore-sized polyethersulfone membrane filters (Supor-200; Pall Corp., East Hills, NY), and frozen for DNA analysis in 250 µL of DNA lysis buffer (20mMTris-HCl pH 8.0, 2mM EDTA pH 8.0, 1.2% v/v Triton X100) [28].

Upon return to the laboratory at the HIMB, coral fragments were rinsed with 1 µm-filtered seawater and placed in a holding tank of 1 µm-filtered seawater maintained at the ambient temperature of 27°C. Four fragments from each colony were mounted randomly in each of eight 0.76mm thick clear vinyl sheets (Figure 1). A sheet was placed in each of eight independently controlled 60 L experimental aquaria. An additional 2-4 extra fragments per colony were also mounted in sheets as backups for fragment mortality. To minimize exposure to disturbance and handling upon removal from the reef, coral samples were collected between 0900 and 0945 and mounted in their final position in the aquaria by 1400 the same day.

All aquaria were filled with 1 µm-filtered, UV-treated seawater, which recirculated through the individual tanks for the duration of the experiment. Each tank was equipped with a full spectrum light (white and UV) on a 12:12 hr light:dark cycle, a protein skimmer, and a submersible pump flowing at 20 gal min-1. The volume of seawater in each tank was monitored daily and salinities kept constant by the addition of sterile, milli-Q freshwater. Temperature and light levels in the tanks were monitored with Onset Computer HOBO temperature and light pendant loggers (UA-002-64 Onset Computer Corporation, Bourne, MA). The loggers were fixed horizontally (light meter facing up) to the vinyl sheets containing coral fragments with cable ties, and the sheets supporting the mounted corals were fixed to the bottom of the tank with suction cups. The seawater temperature was maintained at the ambient temperature of 27°C for 10d to allow corals to acclimate.

After the acclimation period, four tanks were randomly assigned as controls (tanks 1, 8, 9, and 13) and four as treatment (tanks 3, 4, 15, and 16). At 1300 on that day, seawater and coral bacterial communities were sampled, and the seawater temperature in the treatment tanks abruptly increased to 28°C (+1° C over) in treatment tanks. Corals were subsequently sampled from the aquaria at the same time of day (i.e., initiated at 1300 and completed by 1430) after 2, 4, and 6 d of incubation and aquarium seawater after 2 and 7 d. Coral sampling consisted of removing one randomly selected coral fragment per colony per tank and freezing it in liquid nitrogen. Seawater (250 mL) was removed from each tank using acid-washed polycarbonate bottles and subsequently filtered through 25mm diameter Supor-200 membranes (Pall Corp.). Filters were placed in 200 µL of DNA lysis buffer and stored at -80°C until further processed.

T-RFLP of Bacterial SSU rRNA genes: Individual coral fragments were thawed, and a flame-sterilized stainless steel core borer was used to remove three random subsamples. Each subsample consisted of a 6-mm diameter, 6-mm deep core that included the coral tissue, overlying mucus layer, and underlying skeleton. The three subsamples were placed into one sterile bag containing 2 mL of 0.2 μm-filtered 10X Tris EDTA (100 mM Tris, 10 mM EDTA) buffer solution (pH 7.4) and airbrushed with an air gun and sterile pipette tip. The resultant slurry was centrifuged at 19,900 RCF for 30 min at 4 °C. The supernatant was subsequently removed from the sample and the remaining sample pellet was frozen at − 80 °C until processed further. After thawing, genomic DNA was extracted from the coral tissue pellets and seawater filters using the PowerSoil DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA) according to the manufacturer’s protocol. Genomic DNA was eluted in sterile, 0.1 μm-filtered water and stored frozen at − 20 °C. Total genomic DNA yield was assessed on a SpectraMax M2 plate reader (Molecular Device Corp., Sunnyvale, CA, USA) using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen Corp., Carlsbad, CA, USA), prepared according to manufacturer’s protocol.

For terminal restriction fragment length polymorphism (T-RFLP) analysis, the general bacterial primers 27FB- FAM (5-FAM-AGRGTTYGATYMTGGCTCAG-3) and 1492R (5-GGYTACCTTGTTACGACTT-3) were used for the amplification of small subunit ribosomal RNA (16S rRNA) genes from each sample via the polymerase chain reaction (PCR). The MasterTaq System (Eppendorf AG, Hamburg, Germany) was used for all PCR reactions, which were composed of the following (final concentrations): 1X MasterTaq reaction buffer, 2.25 mM Mg²⁺, 0.5X TaqMaster reaction enhancer, 0.2 mM each of the forward and reverse primers, 0.2 mM of each dNTP (Promega, Madison, WI), approximately 160 to 280 ng of genomic DNA template, 2.5 units of MasterTaq DNA polymerase, and sterile water to a final reaction volume of 50 μl. A MyCycler thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) and touchdown protocol were used, which, after a 3 min incubation at 95 °C, consisted of 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. This was followed by 10 cycles of 30 sec at 95 °C, 1min 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. The fluorescently labeled amplicons were purified using the QIAquick PCR purification kit (Qiagen Inc.) following the manufacturer’s instructions. Approximately 100 ng of each purified amplicon was subsequently digested in a 10 μl reaction containing 5 units of HaeIII restriction endonuclease (Promega, Madison, WI) at 37 °C for 6 hours. After purification via gel filtration chromatography with Sephadex G-50 (Amersham Biosciences, Sweden), the restricted samples were adjusted to a final concentration of 35 ng μl⁻¹ and separated via capillary electrophoresis on an automated ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).