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Characterization of the bacterioplankton community in two

contrasting Basque estuaries using massive parallel sequencing
Mikel Aguirre 1, I. Zarraonaindia 2,3, David Abad 1, Aitor Albaina 1, Nestor Etxebarria 4, Andone Estonba 1.
1 Department of Genetics, Physical Anthropology & Animal Physiology. Faculty of Science and Technology. University of the Basque
Country, Spain; 2 Argonne National Laboratory, Argonne, IL 60439, U.S.A.; 3 IKERBASQUE, Basque Foundation for Science, Spain; 4
Department of analytical Chemistry. Faculty of Science and Technology. University of the Basque Country, Spain

Introduction
Planktonic communities are the base of aquatic food webs. Within the organisms composing them, bacteria are the less studied group. The aim of this project is to
characterize the bacterial taxonomic diversity in the planktonic community of two contrasting estuaries: the highly polluted Bilbao estuary and the UNESCO biosphere
reserve estuary of Urdaibai. Although both estuaries have been studied in detail when considering water quality and eukaryotic organisms (Albaina, A.,et al, 2009),
research referring bacterial communities have been traditionally neglected. Therefore, we applied a metabarcoding approach, using the 16S rDNA region of the Earth
Microbiome Project (http://www.earthmicrobiome.org/ & Gilbert et al. 2010). This gene allows us to obtain the highest correspondence between Operational Taxonomic
Units (OTUs) and taxonomic identities due to the huge availability of reference sequences in public databases. Total biodiversity was estimated based on the number of
different OTUs generated by the bioinformatic analysis. Sampling took place monthly, along a salinity gradient during an annual cycle, and a set of environmental
variables (temperature, salinity, dissolved oxygen, heavy metals, ...) were recorded to assess their role in shaping bacterial taxonomic diversity in both estuaries.

Matherial and methods
Our interest is focused on the bacterioplankton community of two contrasting
estuaries from the Bay of Biscay with different levels of anthropogenic pressure:
on the one hand, the Nervion estuary, which was one of the most contaminated
in Europe until the late 80s and now is in a recovery process. On the other hand,
the Urdabai estuary, an UNESCO biosphere reserve. We collected samples along a
salinity gradient ranging from 30 to 35 ‰, monthly throughout a entire year.
Illumina's MySeq platform was applied to sequence 16S amplicon, using Eart
Microbiome Project's protocols, from living organisms between 0.22-20 μm size

range. We have chosen environmental variables such as temperature, pH, heavy
metals and classical organic pollutants (phenols, pesticides, phthalates, ...).
The raw sequences from the Illumina Miseq 2x150 were trimmed using sickle
software. For paired-end merging we used Pear software (J. Zhang et al, 2013).
To perform the taxonomic assignment and the ecological analysis we used the
Quantitative Insights Into Microbial Ecology (QIIME) pipeline (J Gregory Caporaso
et al, 2012) and Greengenes (DeSantis, T. Z. et al, 2006).

Results
Figure A

Figure B

Fresh water

Figure C
Coldest

Winter

Summer

Saltwater

Figure A: PCoA of the distribution of microbial
communities along the salinity gradient.

Hottest

8.49
8.62
8.88
9.17
9.27
9.56
10.14
10.53
10.62
11.05
11.28
11.34
11.44
11.45
11.5
11.58
11.66
11.7
11.79
11.94
12.09
12.14
12.54
12.62
12.65
12.79
14.06
14.49
14.69
15.07
15.27
15.43
15.87
16.24
16.66
16.72
17.05
17.15
17.2
17.28
17.33
17.59
18.63
18.65
19.04
19.13
19.38
19.7
19.78
20.03
20.17
20.18
20.2
20.4
20.41
20.45
20.54
20.62
20.77
20.82
20.91
20.96
21.71
21.78
22.66

Figure C: PCoA of the microbial communities
changes along temperature gradient.

Figure B: PCoA of bacterial communities variations
throughout the seasons.

Figure F

Figure D

Figure E
Figure E: The precipitation effect in heavy metal
concentration. The higher the precipitation, the
lesser the concentration of heavy metals.
Figure D: PCA shows sampling points located in the
graph based on its chemical characteristics during
different seasons: winter (green), spring (ligth blue),
summer (dark blue) and fall (red).

Figure F: PCoA of the changes in microbial
communities with different mercury values.

Figure G
categories
Salinity+Temp+pH
Salinity
Sn+Hg+Cr
Hg

rho statistics
0.52929
0.47273
0.2087
0.20655

p-value
0.05
0.05
0.01
0.01

Figure G: The variables which best explain patterns
in the bacterial communities.

Conclusion
- The physicochemical parameter that mostly influences the microbial communities is the salinity gradient. Other strong parameters are the seasonal effect (temperature
mainly) and pH in the water (Figures A, B, C & G).
- Heavy metal concentrations change along the year (Figures D & F). This may be due to the different amount of precipitation observed at each station. The rainfall
period has a "washing effect" on heavy metals (Figure E). Their concentrations affect lightly to microbial communities in these mesotidal estuaires (Figure G).
- Given that anthropogenic organic contaminants are another factor that can affect planktonic communities, we would perform an analysis of these pollutants in the near
future.

References
- Albaina, A., Villate, F., Uriarte, I., 2009. Zooplankton communities in two contrasting Basque estuaries (1999–2001): reporting changes associated
with ecosystem health. Journal of Plankton Research 31, 739–752.
- DeSantis, T. Z., P. Hugenholtz, N. Larsen, M. Rojas, E. L. Brodie, K. Keller, T. Huber, D. Dalevi, P. Hu, and G. L. Andersen. 2006. Greengenes, a
Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB. Appl Environ Microbiol 72:5069-72.
- Gilbert et al. Meeting Report. The Terabase Metagenomics Workshop and the Vision of an Earth Microbiome Project. Standards in Genomic Science.
(2010) 3:3.
- J. Gregory Caporaso et al. QIIME allows analysis of high-throughput community sequencing data. Nature Methods (2010) doi:10.1038/nmeth.f.303.
- J. Zhang et al. PEAR: A fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics (2014) 30 (5): 614-620.

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