Examined Concentrations
This study focused on three varying concentrations of atrazine commonly found within an oyster's environment. In order to reproduce ecologically relevant concentrations of atrazine within our experiments, data resources overseen by the EPA and USDA were examined and cross referenced with pesticide and herbicide datasets taken by various external sources [9], [11], [14]. Concentrations of atrazine were highest in the upper tributaries of the Chesapeake Bay.
1) 30 µg/L Atrazine: The peak concentration of atrazine detected in any sample within the Chesapeake Bay was 30 µg/L (station L0002488), which is located in a tributary on the Eastern Shore shown in Supplementary Figure 1 . (US EPA 2006)
2) 10 µg/L Atrazine: The general trend in the data indicates that the main detections of atrazine are in the tributaries while significantly lower concentrations have been found in the Bay itself. During June of 2011, atrazine concentrations from the upper Chop Tank River were logged. The average of all recorded concentrations was 0.29 µg/L. The highest recorded concentration was 10 µg/L (Chesapeake Bay Program; USDA: Hively et al. 2011).
3) 3µg/L Atrazine: The EPA’s Maximum Residue Limit (MRL) for atrazine is relevant because it is the limit which the EPA has set forth regulating our drinking water (U. S. EPA MRL).
4) 30µg/L Acetone: Atrazine stock solution was dissolved in a 100% acetone solution. In order to assume continuity throughout the experiment this treatment was added to assure that any noticed effect on development was due solely to atrazine exposure.
The oyster groups spent a total of 3 hours submerged in 2 liters of treated water within each glass tank twice per week on Mondays and Thursdays for X# of weeks in order to mimic the effects of heavy rainfall found within the Chesapeake Bay area (USGS: Chesapeake Region Climate/Precipitation Data). In order to minimize any atrazine contamination of the respective holding tanks after the 3-hour exposure period, each group was washed using a constant stream of pressure-filtered water for three one-minute rinses. Oyster groups were then relocated to their respective 40-liter glass bio-cube tanks. The bio-cube tanks were set to have the same parameters as the large holding tank.
16S Sequencing
A microbiome library was constructed from the tissues of five groups of oysters according to the concentration used in treatments, 30ug atrazine, 10ug atrazine, 3ug atrazine, 30ug acetone, and control group reads were de novo assembled together. The differential expression of microbes was analyzed using the general assembly as a reference for mapping the reads from each condition. In this project, 408,495 pair-end reads were obtained for 16 samples in total, after pair-end reads merging and filtering, 302,751 clean tags were generated, an average of 18,922 clean tags for each sample. Amplicons were performed on a paired-end Illumina HiSeq2500 platform to generate 250bp paired-end raw reads, and then pretreated. Specific processing steps are as follows:
1) Paired-end reads were assigned to a sample by their unique barcode, and the barcode and primer sequence were then truncated.
2) Paired-end reads were merged using FLASH (V1.2.7,http://ccb.jhu.edu/software/FLASH/ ) ,a very fast and accurate analysis tool to merge pairs of reads when the original DNA fragments are shorter than twice of the reads length. The obtained splicing sequences were called raw tags.
3) Quality filtering was then performed on the raw tags under specific filtering conditions of Trimmomatic v0.33 (http://www.usadellab.org/cms/?page=trimmomatic) quality control process. After filtering, high-quality clean tags were obtained.
Statistical Analysis
To characterize microbial communities and to perform functional analyses, the following wrappers have been employed: summarize_taxa_through_plots (to produce the taxonomical files and charts), alpha_rarefaction and beta_diversity_through_plots (to assess respectively the alpha- and beta-rarefaction diversity indices), principal_coordinates.py (to compare groups of samples based on phylogenetic distance metrics). To compare sampling treatments (Control versus all three treatment concentrations) within each sampling season, ANOVA analyses have been performed at genus level (P-value < 0.005; FDR < 0.01) using Excel version 8.13.
Results
Diversity of microbial communities and taxonomic richness
The highest amount of bacterial genera strains identified in this study comprised of Vibrio, Clostridum, and Pseudoalteromonas, while Nocardia, Fusobacterium, Mycobacterium and Planctomycetes were detected at lower levels. The high incidence of Vibrio spp., Clotridium spp., and Pseudoalteromonas spp., may indicate the influence of environmental factors in oyster groups.