Dataset: coral-microbe T-RF - development
Deployment: Rappe_2008

Exposure of P. meandrina embryos and larvae to bacterial cultures. On 21 April 2008, fragments of healthy P. meandrina colonies were removed from the reef flat in Kaneohe Bay, Oahu, HI and held in seawater aquaria for 2 days prior to spawning on the morning of 23 April 2008. Eggs from 10 colonies were combined, fertilized with sperm for 30 min, and subsequently rinsed using seawater sterilized by the method outlined below. The embryos were placed in individual 100-mm-diameter petri dishes containing raw seawater, sterile seawater, or individual bacterial strains as treatments. The bacterial strains utilized in this study are single-strain laboratory cultures that represent some of the major groups of planktonic bacteria present in Kaneohe Bay seawater, as well as an isolate from an adult coral. Water for the sterile-seawater treatment was collected from the surface of Kaneohe Bay 4 days prior to spawning and sterilized by tangential flow filtration (TFF) using a Millipore Pellicon 2 mini-TFF unit equipped with a 30-kDa-cutoff regenerated-cellulose filter cassette (Millipore Corp., Billerica, MA). The sterility of this water was confirmed by microscopy daily. The raw (nonsterile)-seawater treatment was prepared by coarsely filtering Kaneohe Bay surface seawater through a 1.6-um-pore-sized GF/A filter (Whatman International Ltd., Kent, United Kingdom) in order to exclude zooplankton and larger organisms. Bacterial treatments were established by growing bacterial strains in liquid batch culture and subsequently incubating diluted bacterial cells with rinsed coral planulae. After 24 h of incubation, planulae were removed, rinsed, and incubated with the same bacterial strain, freshly diluted. After repeating the treatment again at 48 h and incubating the planulae for another 24-h period (T=72 h), free-living bacterial cells were counted via microscopy (described below) and a subsample collected for community structure characterization. At 170 h, planulae were rinsed and collected for community structure characterization and microscopy analysis, and subsamples of free-living bacterial cells were again collected for characterization of bacterial community structure. This characterization was performed in order to assess whether the bacterial community was altered following incubation with coral embryos.

Axenic cultures of each strain were grown in specific media: Roseobacter strain HIMB1 and SAR11 strain HIMB4 were grown in low-nutrient medium (sterile seawater amended with ammonia and phosphate), Synechococcus strain HIMB12 was grown in low-nutrient medium amended with dilute carbon additions (D-glucose, D-ribose, pyruvate, succinate, ethanol, glycerol and N-acetylglucosamine, each at 0.001% final concentration), and Pseudoalteromonas strain HIMB1276 was grown in R2A medium with seawater as the base (Sigma-Aldrich, St. Louis MO). Cells were grown in 12-h light/dark cycles at 30°C. Every 24 h, 30-ml treatments of each bacterial strain were prepared by diluting growing cultures 10x to 1,000x (e.g., adding 1 to 0.004 ml of culture to 30 ml total) into sterile seawater.

Planulae were rinsed daily using 40-um-mesh-size cell strainers (Fisher Scientific, Pittsburg, PA) and sterile seawater and placed into new petri dishes containing fresh treatments. Treatments were initially replicated in triplicate, but after high embryo mortality during the first 24 h, replicates were subsequently combined to ensure that adequate material was available for the duration of the experiment. After 72 h (i.e., following three 24-h treatments), the abundance of microbial cells present in the treatment water exposed to the planulae was determined by microscopic enumeration as described above. Additionally, after 72 and 170 h (i.e., following three and seven 24-h treatments, respectively), subsamples of 30 to 50 ml of treatment water were filtered onto 13-mm-diameter, 0.2-_mpore- size polyethersulfone membrane filters (Supor 200; Pall Gelman, Inc., Ann Arbor, MI). Filters were stored at_80°C inDNAlysis buffer (20 mM Tris-HCl [pH 8.0], 2 mM EDTA [pH 8.0], 1.2% [vol/vol] Triton X-100) for subsequent DNA extraction. After 170 h, 100 to 200 planulae per treatment were preserved for microscopy and frozen to -20°C as described above, and 50 to 100 planulae per treatment were preserved in 250 ul of lysis buffer and frozen to -80°C for subsequent DNA extraction.

T-RFLP of bacterial SSU rRNA genes. DNA was extracted using the DNeasy tissue kit (Qiagen, Inc., Valencia, CA) with modifications and quantified using the PicoGreen fluorescent assay (Invitrogen Corp., Carlsbad, CA) on a SpectraMax M2 plate reader (Molecular Devices Corp., Sunnyvale, CA). For terminal restriction fragment length polymorphism (T-RFLP) analysis, bacterial small-subunit (SSU) rRNA genes were amplified via PCR using oligonucleotide primers 27F-B-FAM (5’-AGRGTTYGATYMTGGCTCAG-3’) and 519R-VIC (5’-GWATTAC CGCGGCKGCTG-3’), with “FAM” and “VIC” indicating 5’-end labeling with FAM or VIC fluorochromes, respectively. Each 50-ul PCR mixture contained 2 U of Sahara enzyme (Bioline USA, Inc., Taunton, MA), 1x Sahara reaction buffer, 2 mM Sahara MgCl2, 200 uM each deoxynucleoside triphosphates (dNTPs), 200 nM each primer, and 100 ng of template genomic DNA (up to 1 ug for samples that did not amplify at lower concentrations). After an initial denaturation step at 95°C for 5 min, the reaction conditions were as follows: 29 cycles of 95°C denaturation for 30 s, 55°C annealing for 1 min, and 72°C extension for 2 min, concluding with an extension at 72°C for 20 min. The reactions were performed in a MyCycler personal thermal cycler (Bio-Rad Laboratories, Hercules, CA). Products were purified using the QIAquick PCR purification kit (Qiagen, Inc.) and subsequently restricted in a 10-ul reaction mixture containing 100 ng of purified amplification product, 2 ug of bovine serum albumin (BSA), 1x enzymatic reaction buffer, and 5 units of HaeIII restriction endonuclease (10 units per ul; Promega, Madison, WI) for 7 h at 37°C. Restriction digests were purified using the QIAquick nucleotide removal kit (Qiagen, Inc.).