Examined Concentrations
This study focused on four 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 (USDA 2006; Flood et al. 2015; Powell et al. 2017; Hively et al. 2011; EPA 2017). Concentrations of atrazine were observed to be highest in the upper tributaries of the Chesapeake Bay. The following outlines the chosen concentrations and the reasoning for choosing the concentration.
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) 20 µg/L Atrazine: A half-way point in between 30 µg/L Atrazine and 10 µg/L Atrazine for data exploration purposes.
3)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).
4) 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).
5) 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.
16S Sequencing
As stated previously, two separate sequencing events took place during this study. The first was conducted on specimens from the first stabilization and treatment periods. The second sequencing event was conducted in specimens from the second stabilization and treatment periods. The first sequencing event included five examined concentrations: 30 µg/L Atrazine, 10 µg/L Atrazine, 3µg/L Atrazine, 30µg/L Acetone and a control and included a total of 16 samples (six 30µg/L Acetone, three controls, three 30µg/L atrazine, two 10µg/L and two 3µg/L. Specimens in each group were sequenced both directly following the treatment periods and following a 30-day recovery period. The second sequencing event included six examined concentrations: 30 µg/L Atrazine, 20 µg/L Atrazine, 10 µg/L Atrazine, 3µg/L Atrazine, 30µg/L Acetone and a control and included a total of 24samples (four per sample). Amplicons were performed on a paired-end Illumina HiSeq2500 platform to generate 250bp paired-end raw reads, and then pretreated. Specific processing steps for both sequencing events were completed 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 were 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 treatment concentrations) within each sampling season, ANOVA analyses have been performed at genus level (P-value < 0.005; FDR < 0.01) using Excel version 16.13.
Results
First sequencing event:
Diversity of microbial communities and taxonomic richness
Of the top ten most abundant bacterial genera strains identified from the first sequencing event, Vibrio, Clostridum, and Pseudoalteromonas were detected at high levels, while Nocardia, Fusobacterium, Mycobacterium and Planctomycetes were detected at lower levels. The high incidence of Vibrio, Clostridium, and Pseudoalteromonas species may indicate the influence of environmental factors in oyster groups. Additionally, a variety of unknown, likely transient, genera were also detected across all samples, henceforth these genera will be referred to as "others".