Abstract:
Marine sponges are remarkable filter feeders (up to 24,000 L of water kg-1 day-1) and can strongly influence the water quality of their environment. Many species harbor a high diversity and abundance of microbes (Archaea, Bacteria and Eukaryotes) which constitute up to 40% of the total sponge biomass. Bacteria abundance in sponge tissues can be two- to four-fold higher than concentrations in the surrounding seawater. Furthermore, some Bacteria permanently inhabit the sponge tissues while others are rapidly consumed. Culture-independent techniques have shown these sponge-specific bacterial assemblages are diverse, including 14 phyla of Bacteria. We investigated diversity of Bacteria within Cinachyrella sp. tissues from a) specimens directly collected from the environment and b) specimens kept in tanks for up to 3 weeks after collection by 16S rDNA clone libraries and tag pyrosequencing. Results showed that Cinachyrella sp., an important component of South Florida reefs and benthic ecosystems, proved to be a good model sponge. Many Bacteria present in the sponge tissues were rarely detected from the surrounding seawater. The most common Bacteria associated with Cinachyrella sp. were most closely related to the taxa Chloroflexi and Actinobacteria. These results will have important implications for a larger study on the effects of the Deepwater Horizon oil spill on marine sponges’ symbionts.
Suggested Citation:
Marie Cuvelier, Rebecca Vega Thurber, Jose V Lopez. 2014. 16S rRNA-based microbial community profile of model sponge, Cinachyrella sp. from the Broward County Florida reefs, 2011.. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7R49NRR
Data Parameters and Units:
rDNA sequences with and without singlets (taxonomy and OTU number). Environmental gene sequencing by cloning the specific 16S rRNA gene to produce a profile of microbial community diversity in Cinachyrella sp. sponges described in Operational Taxonomic Units (OTUs). Group 1. specimens directly collected from the environment, Group 2. specimens kept in tanks for up to 3 weeks after collection by 16S rDNA clone libraries and tag pyrosequencing, Most abundant OTUs in Seawater, Cinachyrella Group 1, Cinachyrella Group 2.
Methods:
Sampling Cinachyrella sp. (class Desmospongiae) sponges were collected by SCUBA diving at Dania Beach reef, Broward County, Florida, USA (latitude and longitude 26.051425 N, 80.112141 W) in a water depth of 6 m. Individual sponges were cut at the base with a dive knife and placed in individual Nasco Whirl Pak bags filled with ambient seawater. DNA extraction About ¼ of a sponge was used for genomic DNA extraction. The piece was placed in a sterile petri dish and the ectoderm (darker outer layer) was immediately removed using a sterile scalpel. The sample was transferred to a new petri dish and 5 ml of L buffer (10 mM Tris pH=7.6, 100 mM EDTA, 20 mM NaCl) was added. The sponge was minced into very fine pieces in the buffer and all the cell suspension was collected into 1.7 mL tubes. The samples were then centrifuged for 15 min at 16,000 g at 4 °C. The supernatant was decanted and the pellets were transferred to beads tubes and extracted using the MO BIO PowerSoil DNA isolation kit according to the manufacturer’s instructions (MO BIO, Carlsbad, CA). PCR and analysis Approximately 291 bp of the 16S rRNA gene was amplified by PCR using the universal bacterial and archaeal primers: 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) (Caporaso et al., 2010 PNAS) which contained a unique barcode used to Tag each PCR product. This primer set was chosen because it targets a broad range of bacterial and archaeal taxa, with the exception of a few groups (Bates et al., 2010, Caporaso et al., 2010. PCR consisted of 2 reactions of 30 µL with (for each reaction): 3 µL of each forward and reverse primer (10 µM), 1 µL of template DNA, 3 µL of buffer, 3 µL of MgCl2 (25 mM), 2.4 µL of dNTPs (2.5 mM), 0.15 µL of Taq (High Fidelity Taq, TaKARa Otsu, Shiga, Japan). Thermal cycling was initiated with an initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation for 45 s at 94 °C, annealing for 60 s at 50 °C and elongation at 72 °C for 90 s and a final extension step for 10 min at 72 °C. 5 µL of PCR products were visualized on a 1.5% agarose gel (containing Gel Red). Sequences were analyzed using QIIME version 1.6 (Caporaso et al., 2010 Nature Methods) and CloVR 1.0-RC4 (Angiuoli et al., 2011 BMC bioinformatics). Only sequences with an average quality score >25 and of length greater were used in the analysis. Operational Taxonomic Units (OTU) were picked using the Uclust method in QIIME and sequences with >97% identities were considered to be within the same OTU.
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