Discussion
    Results from studies employing molecular methodologies, including NGS, have found that Proteobacteria and Cyano- bacteria are abundant members of bivalve gut communities, regardless of host species (King et al. 2012, Trabal et al. 2014). Cyanobacteria, however, are likely derived from the particle diet, and not long-term residents of the gut community (Pierce 2016). Trabal et al. (2014) depurated oysters before microbial community characterization and did not observe Cyanobacteria represented in any of the threeCrassostrea spp. analyzed. In individual oysters, Proteobacteriamay account for >50% of the total abundance, with Alpha (a)- and Gamma (g)-proteobacteria consistently reported as the most abundant (Pujalte et al. 1999, Hernandez-Zarate & Olmos-Soto 2006, Green & Barnes 2010, Fernandez-Piquer et al. 2012, Trabal et al. 2014, Wang et al. 2016, Lokmer et al. 2016b, Rong et al. 2018, Pierce & Ward in review). Within the g -Proteobacteria,Shewanella, Pseudoaltermonas, Oceanospiralles, and Vibrio are major taxa reported in this study. Other dominant phyla identified included those seen by a variety of studies examining microbiomes in bivalves: Tenericutes, Bacteroidetes, Actino- bacteria, Firmicutes, Chlamydiae, Fusobacteria, Spirochaetes,Chloroflexi, Plantomycetes, and Verrucomicrobia (Fernandez- Piquer et al. 2012, King et al. 2012, Trabal et al. 2012, Trabal et al. 2014, Cleary et al. 2015, Roterman et al. 2015, Arfken et al. 2017).
     These results indicate that oysters maintain similar microbial communities over time, acting as a host for a specific set of core microbiota. The study found that control groups maintained a common community of microbes in their overall body system throughout the experiemnt. Conversely, we observed significant compositional variation between and within experimental groups in the genus PseudoalteromonasVibrio, and Nocardia. The presence of the genus Nocardia is of particular interest as they are known oyster pathogens which cause round yellow-to-green pustules up to 1 cm in diameter to be displayed on the surface of the mantle, gill, adductor muscle, and heart (Friedman 1998). Nocardia were found only within the atrazine treated groups. During the 30-day exposure period no significant relative abundance was observed for Nocardia in all three atrazine exposed treatment groups, however, after the 30-day rest period a significant relative abundance was detected. This suggests that atrazine selected for the subsequent survival and colonization of Nocardia. Conversely, Psuedoalternamonas, Clostridium and fusobacterium species were found in lesser degree within atrazine treated samples when compared to the controls.
This finding indicates that atrazine exposure may be play a role in facilitating an environment which does not allow for Psuedoalternamonas, Clostridium and/or fusobacterium species to colonize oysters effectively.  
    Interestingly, populations of bacterial species which decreased in the presence of atrazine continued to sustain low abundance % after a 30-day rest period, indicating that atrazine may have lasting effects (greater than 1 month) in the population dynamics of C. virginica. Bacterial species known to be pathogenic to C. virginia and thus related to immunity and defense were of particular interest in order to uncover how atrazine is affecting the molecular-bacterial basis of oyster tolerance/resistance to the herbicide’s toxic effects. As has been reported in other studies (Westlund et. at., 2017; Romero et. al., 2002; A Arfken et. al., 2017;  Pierce 2016) our results confirm that oysters maintain similar microbial communities over time. The health of C. virginica is impaired by inflowing storm-water runoff from nearby agricultural fields, which carry large amounts of atrazine into estuarine waters, however,  the knowledge of oyster-associated bacterial community response to biocidal runoff is scarce.
The sequence data obtained could be used to compare oyster aquaculture management strategies as well as aquaculture practiced in different regions that may have similar or different climactic conditions. It is also meant to facilitate a greater understanding of how atrazine, as a persistant environmental condition effects oyster-prokayote interactions. Further studies of this nature could reveal important links between oyster farming, environmental factors, husbandry strategies, as well as legal regulations currently governing the surrounding areas of the Chesapeake Bay.  
Future work:
Many of the mechanisms that mediate prokaryote–host symbioses are unknown or unclear. Both extrinsic and intrinsic factors are at play, with no clear dominant influence. The po- tential for gut microbial communities to effect bivalve digestive enantiostasis and pathogen accumulation is great. Thus, un- derstanding the natural spatial and temporal variation of these communities, the influence of the surrounding seawater and particulate-associated microbes, and the impact that dis- turbances in the microbiome have on bivalve enantiostasis is imperative. Building on this work will allow more direct probing of questions relating to the role of microbial communities in host physiological functioning and enantiostasis. The complex relationships between the envi- ronment, bivalves, and maintenance of their microbial communities have only begun to be probed in the past 30 years. Although this study and many others have contributed important information, an understanding of whether the host, the resident microbial communities, or both produce critical di- gestive enzymes is nascent, and many questions remain. For example, one unknown is the contribution of enzymes from lysed cells, which has been shown to impact digestion even after bacteria are eradicated (Harris 1993). A second unknown is the contribution of bacterial–bacterial or bacterial–animal hori- zontal gene transfer. Several examples within sessile marine invertebrate phyla have emerged to suggest that the in- corporation of bacterial genes into host animal genomes occurs and is related to improving metabolic function (Boto 2014, Degnan 2014). In this case, bacterial genes responsible for en- zyme production and other metabolic processes could be transferred to the eukaryotic host genome. Finally, functional redundancy of enzyme production genes could occur within the microbial community. As such, the antibiotic elimination of certain bacteria may not result in an overall disruption of en- zyme production.
As with most research, results only generate more questions. There are many more topics to examine relating to the bivalve microbiome. For example, do core microbiome trends hold true across bivalve species? Do they hold true with other suspension feeders (e.g., Crepidula spp.)? Are observed similarities between oyster and mussel microbiomes a result of their shared feeding mechanism? Are observed differences between oyster and mussel microbiomes a result of their genetic differences? Do other marine filter feeders harbor the same numbers and types of bacteria as bivalves? Do they share a core? With continued reference to the eastern oyster, do Crassostrea virginica from other locations (i.e., Chesapeake Bay and Gulf of Mexico) harbor the same bacteria as those in Long Island Sound? How far do spatial trends extend until they are broken? Does the environment impart more of an influence in some locations than in others? Do the core microbiota contribute to specific host physiological functions? Does a high diversity in the gut microbiome inhibit pathogen colonization in the long term? Investigating such questions would be beneficial, resulting in an enhanced understanding of bivalve host–bacterial interactions.
Conclusion
    Understanding natural variation in the genetic and func- tional diversity of the oyster-associated microbial communities is vital for establishing a baseline to which the effects of extrinsic and intrinsic factors can be compared. The presence of atrazine in the Chesapeake Bay may be selecting for pathogenic bacterial groups to reside within the oysters of the Chesapeake Bay and its tributaries. The effects of this compositional shift remain unclear, therefore, future research efforts should focus on understanding the relationship between biocidal herbicides and oyster-prokaryote interactions.  Extended experiments with more time points, as well as repeated challenges, would help to further elucidate the role of oyster-associated microbial communities to the overall physiological functioning of the host. This research provides a vital baseline for future research aimed at understanding the role gut microbes have in oyster physiology. \(\)
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  \(\)Acknowledgements
We would like to thank Horn-point laboratory for providing the spat used throughout the experiment. Dr. Tara Scully for acting as the lead investigator for the project. The George Washington University, for housing the project, and providing the funds necessary for it to be carried out. And finally, to each of the authors for their hard work and perseverance. 
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