Dataset: coral life stages - Illumina
Deployment: Rappe_2010

Fungia scutaria sample collection

Adult specimens of the solitary coral F. scutaria were collected from Kaneohe Bay on the northeastern shore of Oahu, Hawaii in the Pacific Ocean in June 2010 and kept in open-system tanks (sea tables) at the Hawaii Institute of Marine Biology in anticipation of spawning events. Following spawning, eggs and sperm were collected for fertilization in Petri dishes, and replicate batches of larvae were raised in 0.1 µm-filtered (sterile), 1.6 µm-filtered (excludes Symbiodinium and zooplankton), 20 µm-filtered (excludes zooplankton) or raw seawater. Samples from each life history stage (eggs, larvae, juveniles, and adults) were frozen for DNA analysis in DNA lysis buffer. Eggs and embryos were rinsed in sterilized seawater upon sampling. Each adult sample consists of three small pieces of an individual coral combined in one tube. Ca. 1 L of seawater was sampled from the sea tables one hour prior to the final spawning event, filtered, and frozen for DNA analysis in DNA lysis buffer. 50 mL of the culture water was sampled from the Petri dish treatments (originally 0.1 µm-filtered, 1.6 µm-filtered, or 20 µm-filtered seawater) after approximately 72 hours of exposure to the developing coral larvae during the final experiment.

Montipora capitata sample collection

Fragments of adult specimens of the colonial coral M. capitata were collected from Kaneohe Bay in June 2010 and July 2011 and kept in sea tables at HIMB in anticipation of spawning events. Following spawning, egg-sperm bundles were collected from the sea tables for fertilization in Petri dishes, and replicate batches of larvae were raised in 0.1 µm-filtered, 1.6 µm-filtered, or 20 µm-filtered seawater. Samples from each life history stage (egg-sperm bundles, larvae, juveniles, and adults) were frozen for DNA analysis in DNA lysis buffer. Egg-sperm bundles were rinsed in TFF sterilized seawater upon sampling. Each adult sample consists of three small pieces of an individual coral combined in one tube. Ca. 1 L of seawater was sampled from the sea tables approximately one hour prior to the final spawning event, filtered, and frozen for DNA analysis in DNA lysis buffer. 35 mL of the culture water was sampled from the Petri dish treatments (originally 0.1 µm-filtered, 1.6 µm-filtered, or 20 µm-filtered seawater) after approximately 72 hours of exposure to the developing coral larvae during the final experiment in 2010, filtered, and frozen for DNA analysis in DNA lysis buffer. Additionally, throughout summer 2010, field-spawned egg-sperm bundles and field-fertilized larvae were collected from Kaneohe Bay and raised under the same treatments (0.1 µm-filtered, 1.6 µm-filtered, 20 µm-filtered, or raw seawater), and sampled in the same manner and at the same life-history stages (egg-sperm bundles, larvae, juveniles, and adults) as the corals spawned in sea tables. Ca. 1 L of seawater was sampled from the Kaneohe Bay lagoon 1 hour prior to the August 2010 field-spawning event and 1 hour after the June and July 2010 field-spawning events, filtered, and filters were frozen for DNA analysis in DNA lysis buffer.

DNA extraction and Illumina amplicon sequencing of bacterial SSU rRNA genes 

Nucleic acids were extracted from F. scutaria corals and filters using the NucleoSpin Soil Kit (Macherey-Nagel, Duren, Germany) following the manufacturer’s protocol, modified as follows: lysis buffer SL1 and Enhancer SX were used, lysis time was increased to 20 min and two subsequent elution steps were performed with 50 µl fresh elution buffer each. DNA quality and yield were checked by agarose gel electrophoresis.

Nucleic acids were extracted from M. capitata samples and filters using the PowerSoil DNA Isolation Kit (MO BIO, Carlsbad, CA) following the manufacturer’s protocol, modified as follows: initial physical disruption time (via vortex) was increased to 30 min, and two subsequent elution steps were performed with 100 µl fresh DNA-Free PCR Grade water each. DNA quality and yield were checked by agarose gel electrophoresis.

The V4 region of the 16S rRNA gene was amplified with the primer pair F515/R806, which include Illumina flowcell adapter sequences and a sample-specific barcode on the reverse primer. The MasterTaq Kit (5 PRIME, Hamburg, Germany) was used for all PCR reactions. Amplification was performed using a touchdown protocol as in as follows: initial denaturation at 95°C for 3 min, and 30 cycles at 95°C for 30 sec, 65°C for 1 min (decreasing by 0.5°C per cycle), and 72°C for 2 min. After these 30 cycles, a further 5 – 7 cycles were performed using an annealing temperature of 50°C, and concluding with a final extension at 72°C for 20 min. Resulting PCR amplicons were quantified using the PicoGreen dsDNA assay (Invitrogen, Carlsbad, CA), pooled at equimolar concentrations and subsequently cleaned using the UltraClean PCR Clean-Up Kit (MO BIO Laboratories, Carlsbad, CA). Sequencing of the F. scutaria PCR products was performed on an Illumina MiSeq at the University of Colorado BioFrontiers Institute (Boulder, CO), while sequencing of the M. capitata PCR products was performed on an Illumina MiSeq at the University of Hawaii HIMB Genetics Core Facility (Kaneohe, HI).