Suggested Citation:
Kamalanathan, Manoj. 2021. Microcosm: Flask experiment of phytoplankton-bacteria interaction in a natural community using control and water accommodated fraction (WAF) treatments; non-radioactive experiment, 16s sequencing. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-vtz2-9f39
Purpose:
As microbial interactions are an important driver in the formation of marine snow, exposure to oil is bound to alter their interactions and therefore the marine ecosystem dynamics. Therefore, the aim of our study was to characterize microbial interactions and show how they are impacted by oil exposure, by microbial community composition (16S and 18S rRNA gene sequencing) analysis.
Methods:
We elucidated the interactions between phytoplankton and bacteria in oil and in seawater with no additions by subsampling the natural seawaters from the Gulf of Mexico with their ambient communities. The phytoplankton and bacteria assemblages were revealed through 18S and 16S rRNA sequencing throughout the experiments. PCR amplification was performed using Platinum Taq DNA Polymerase (ThermoFisher Scientific) followed by the 16S/18S rRNA gene Illumina amplicon protocol from the Earth Microbiome project. Samples were amplified in triplicate (25 μL reactions) using the following cycling parameters: 95°C for 3 minutes, 30 cycles of 95°C for 45 seconds, 50°C for 60 seconds, and 72°C for 90 seconds, and a final elongation step at 72°C for 10 minutes. Amplifications were performed using the modified 515F-806R primer pair (10μM each) that reduce bias against the Crenarchaeota and Thaumarchaeota lineages as well as the SAR11 bacterial clade for the prokaryotes (Apprill et al., 2015 and Parada et al., 2016). Additionally, the primer pair was modified to include Golay barcodes and adapters for Illumina MiSeq sequencing. Details of the final primer sequences can be found in Walters et al. (2015). For the eukaryote analysis, the primer pair utilized was V8f-1510r (Bradley et al., 2016). The V8V9 hypervariable region on the 18S rRNA for eukaryotes and the V4 hypervariable region on the 16S rRNA for prokaryotes were used for amplification (Bradley et al., 2016 and Caporaso et al., 2011). The triplicate products were combined together following amplification and run on a 1.5 % agarose gel to assess the success of amplification and relative band intensity. Triplicate samples of amplicons were then pooled and purified using the Qiagen PCR Clean-up kit (Qiagen, USA). 50 ng of each sample was pooled and the purified library, along with aliquots of the three sequencing primers, were sent to the Texas A&M Genomics and Bioinformatics Services (College Station, Texas, USA) for MiSeq sequencing (v2 Nano chemistry, 2 × 250 bp). Sequence reads (16S rRNA and 18S rRNA) were processed separately using mothur v.1.39.5 following the MiSeq SOP (https://www.mothur.org/wiki/MiSeq_SOP) (Kozich et al., 2013 and Schloss et al., 2009). Vegan package was used to calculate the diversity and richness indices (Observed richness, Simpson, Shannon, Effective diversity).
Provenance and Historical References:
Apprill Amy, Sean McNally, Rachel Parsons, and Laura Weber. 2015. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquatic Microbial Ecology 75(2):129-37. DOI:10.3354/AME01753
Bradley Ian M., Ameet J. Pinto, and Jeremy S. Guest. 2016. Design and evaluation of Illumina MiSeq-compatible, 18S rRNA gene-specific primers for improved characterization of mixed phototrophic communities. Applied and Environmental Microbiology 82(19):5878-5891. DOI: 10.1128/AEM.01630-16
Caporaso J. Gregory, Christian L. Lauber, William A. Walters, Donna Berg-Lyons, Catherine A. Lozupone, Peter J. Turnbaugh, Noah Fierer, and Rob Knight. 2011. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences. 108(Supplement 1):4516-4522. DOI: 10.1073/pnas.1000080107
Kozich James J., Sarah L. Westcott, Nielson T. Baxter, Sarah K. Highlander, and Patrick D. Schloss. 2013. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and Environmental Microbiology. 79(17):5112-5120. DOI: 10.1128/AEM.01043-13
Parada Alma E., David M. Needham, and Jed A. Fuhrman. 2016. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environmental Microbiology 18(5):1403-1414. DOI: 10.1111/1462-2920.13023
Schloss Patrick D., Sarah L. Westcott, Thomas Ryabin, Justine R. Hall, Martin Hartmann, Emily B. Hollister, Ryan A. Lesniewski, Brian B. Oakley, Donovan H. Parks, Courtney J. Robinson, Jason W. Sahl, Blaz Stres, Gerhard G. Thallinger, David J. Van Horn, and Carolyn F. Weber. 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology 75(23):7537-41. DOI: 10.1128/AEM.01541-09
Walters William, Embriette R. Hyde, Donna Berg-Lyons, Gail Ackermann, Greg Humphrey, Alma Parada, Jack A. Gilbert, Janet K. Jansson, J. Gregory Caporaso, Jed A. Fuhrman, Amy Apprill, and Rob Knight. 2015. Improved bacterial 16s rRNA gene (V4 and V4-5) and fungal transcribed spacer marker gene primers for microbial community surveys. mSystems 1(1): e0009–15. DOI: 10.1128/mSystems.00009-15
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