Bacterial in the Amundsen Sea Polynya (Southern Ocean): community composition in environmental samples and mesocosm experiment

Versões

A tabela abaixo mostra apenas versões de recursos que são publicamente acessíveis.

Como citar

Pesquisadores deveriam citar esta obra da seguinte maneira:

Richert I, Dinasquet J, Logares R, Riemann L, Yager P, Wendeberg A, Bertilsson S (2019): Bacterial in the Amundsen Sea Polynya (Southern Ocean): community composition in environmental samples and mesocosm experiment. v1.1. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=bacterial_diversity_admunsen_sea_southern_ocean&v=1.1

Direitos

Pesquisadores devem respeitar a seguinte declaração de direitos:

O editor e o detentor dos direitos deste trabalho é SCAR - Microbial Antarctic Resource System. This work is licensed under a Creative Commons Attribution Non Commercial (CC-BY-NC 4.0) License.

GBIF Registration

Este recurso foi registrado no GBIF e atribuído ao seguinte GBIF UUID: fe6e1a81-7a45-45b2-8faa-f164647b8684.  SCAR - Microbial Antarctic Resource System publica este recurso, e está registrado no GBIF como um publicador de dados aprovado por Scientific Committee on Antarctic Research.

Palavras-chave

Metadata

Contatos

Cobertura Geográfica

Amundsen Sea Polynya, Southern Ocean, Antarctica

Cobertura Taxonômica

Bacteria 16S ssu rRNA gene

Cobertura Temporal

Dados Sobre o Projeto

Nenhuma descrição disponível

O pessoal envolvido no projeto:

Métodos de Amostragem

Seawater was collected in 12 L Niskin bottles attached to a 24-bottle SBE 32 rosette; coupled to the rosette was a system of sensors reading depth-resolved pro les of temperature [° C], conductivity [S m−1], oxygen [mg L−1], photosynthetically active radiation (PAR) [μmol photons s−1 m−2 ] and uorescence [mg m−3 chl-a] for each cast (SBE 911, Sea-Bird Electronics, Bellevue, Washington, USA). Water samples for incubation experiments were processed immediately at 2°C in a temperature-controlled room.

Descrição dos passos do método:

1. Processing of the samples used in the mesocosm experiments: To assess bacterioplankton responses to light and dark conditions in the absence of larger predators and eukaryotic phytoplankton, 0.2 μm filtered seawater in 1 L acid washed polycarbonate bottles was inoculated with 5% [v/v] 0.6 μm filltered seawater from the same depth using a vacuum pump to a total volume of 1 L. The experimental design by station included two factors (water mass source of inoculum: epipelagic and mesopelagic) with two treatments (dark and light) for each inoculum. Triplicate incubations were conducted under the dark and light conditions using water from each of three stations: 35 (73°27′95′′S, 112°10′41′′W ), 50 (73° 41′60′′S, 115°25′03′′W ) and 57.2 (73°70′73′′S, 113°26′5′′W ). Each experiment included one inoculum from the light-exposed AASW, and one from the mesopelagic zone from either WW or mCDW. Each 0.2-μm ltered seawater medium came from the same depth and station as its inoculum. The light source imitated light levels at approximately 20–50 m below the surface (Philips TLD-18W/18 blue, 1.5–1.99 *10−2 μmol photons s−1 m−2) in a PAR range of 400–500 nm (according to manufacturer), excluding the short-wavelength UV. After a 7-day incubation at near in situ temperature (0.5°C), bacteria from the full sample volume were collected by vacuum filtration onto 0.2-μm, 47-mm Supor filters (Pall, Lund, Sweden) and stored at −80°C in sucrose lysis buffer (20% sucrose, 50 mM EDTA, 50 mM TrisHCl, pH = 8). Darkness was achieved by covering the bottles with black and lightproof foil.
2. The DNA was extracted using a phenol-chloroform extraction approach as previously described (Riemann et al., 2000). Prior to extraction, microorganisms were enzymatically digested for 30 min with lysozyme at 37°C followed by an overnight digestion with Proteinase K (both 20 mg ml−1, Sigma Aldrich) at 55°C (Boström et al., 2004). The 16S rRNA genes were amplified using the bacterial primers Bakt_341F (CCTACGGGNGGCWGCAG) and Bakt_805R (GACTACHVGGGTATCTAATCC) with 454-Lib-L adapters and sample-specific barcodes on the reverse primer (Herlemann et al., 2011). Each set consisted of up to 72 samples with individual barcodes pooled for sequencing.Triplicate PCR reactions for each sample were carried out with 10 to 70 ng extracted environmental DNA as template. Each 20-μl reaction also contained Phusion Hot Start high- delity DNA polymerase ( thermo Scientific). Ampli cation was carried out by initial denaturation at 98°C for 30 seconds followed by 25 cycles of an initial 98°C denaturation for 30 seconds, subsequent annealing at 50°C for 30 seconds and 30-second extension at 72°C. ese 25 cycles were followed by a final 7-min extension at 72°C. Triplicate reactions for each sample were pooled and PCR products were purified using the Agencourt AMPure XP kit according to manufacturer instructions (Beckman Coulter) and quantified with a Picogreen quantification essay (Invitrogen). Equimolar amounts of amplicon from each sample were pooled and sequenced by 454 pyrosequencing using Titanium chemistry at the SNP/SEQ SciLifeLab platform hosted by Uppsala University (Sweden).

Citações bibliográficas

1. Richert, I., Dinasquet, J., Logares, R., Riemann, L., Yager, P. L., Wendeberg, A., & Bertilsson, S. (2015). The influence of light and water mass on bacterial population dynamics in the Amundsen Sea Polynya.

Metadados Adicionais