Discussion
Results from studies employing molecular methodologies, including NGS, have found that Proteobacteria and Cyanobacteria are abundant members of bivalve gut communities, regardless of host species (King et al. 2012, Trabal et al. 2014). 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, Pseudoaltermonas, and Vibrio are major taxa reported in this study. Other dominant phyla identified included acteroidetes, Actinobacteria, Firmicutes, Chlamydiae, fusobacteria, spirochates, chroloflexi, 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).
Achromobacter, Aeromonas, Flavobacterium, Micrococcus, and Vibrio are bacterial genera commonly isolated from bivalves (Colwell & Liston 1960, Vasconcelos & Lee 1972, Pillai 1980, Kueh & Chan 1985, Olafsen et al. 1993, Pujalte et al. 1999). More recent culture-independent work has shown that although these genera are commonly present, they do not necessarily represent most of the community (Romero et al. 2002, Winters et al. 2010, Wegner et al. 2013, Trabal et al. 2014, Li & Wang 2017). Our results indicate that oysters act as a host for a specific set of core microbiota.
From the PCoA plot (Fig. 3). we can ascertain that ascertain that the control group’s biological replicates are maintaining a more common community of microbes relative to the other treatment groups. Between all treatment groups, we observed significant compositional variation between and within experimental samples in the genus Pseudoalteromonas, Vibrio, and Nocardia. Nocardia spp. were found only within the atrazine treated groups. This suggests that atrazine selected for the subsequent survival and colonization of Nocardia spp. The presence of the genus Nocardia was 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). Psuedoalternamonas, fusobacterium and Clostridium species were found to a lesser degree within atrazine treated samples when compared to the controls. This finding indicates that atrazine exposure may be play a role in crafting an environment which does not allow Psuedoalternamonas, Clostridium and/or fusobacterium species to colonize oysters effectively.
Currently, there are very few studies of probiotic bacteria in bivalve molluscs – almost none in C. virginica – and the protective role played by bacteria is poorly understood. However, the majority of these studies have focused on the protective role of putative probiotic bacteria against Vibrio pathogens (Sakawski 2015). For example, Alteromonas haloplanktis isolated from scallops displayed inhibition against pathogens Vibrio anguillarum and Vibrio alginolyticus in vitro (Riquelme, Hayashida et al. 1996). Similarly, an unidentified bacterium S21 isolated from rearing seawater of C. gigas increased larval survival from 8.4% to 78% after 24 hours of exposure to V. alginolyticus (Nakamura, Takahashi et al. 1999), and Aeromonas media A199 protected against Vibrio tubiashii (Gibson, Woodworth et al. 1998) through indole production (Lategan, Booth et al. 2006). This is of note since 20% of bacterial isolates from C. virginica were capable of producing indole (Murchelano and Brown 1968), and bacteria isolated from C. virginica significantly improved survival of larvae challenged with Vibrio corallilyticus (Lim, Kapareiko et al. 2011). C. virginica inner shell bacterial isolate Phaeobacter sp. S4 also protected C. virginica larvae and juveniles challenged with R. crassostreae and V. tubiashii (Karim, Zhao et al. 2013). However, the protection provided by these probiotics was short-lived (Karim, Zhao et al. 2013), and more work is necessary to understand the protective potential of the commensal community in C. virginica larvae and adults.
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, their genetic differences, or both? 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 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 functional 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, as such, future research efforts should focus on understanding the relationship between commonly used biocidal substances 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.
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 potential for herbicide runoff to effect bivalve digestive enantiostasis and pathogen accumulation is great. Thus, understanding the natural spatial and temporal variation of these communities, the influence of the surrounding seawater and particulate-associated microbes, and the impact that disturbances 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 environment, 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 to this growing body of literature, an understanding of whether the host, the resident microbial communities, or both provide critical 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 horizontal gene transfer. Several examples within sessile marine invertebrate phyla have emerged to suggest that the incorporation 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 enzyme 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. Therefore, the elimination of certain bacteria may not result in an overall disruption of enantiostasis.
The sequence data obtained for this study 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 persistent environmental condition effects oyster-prokayote interactions. Further studies of this nature could reveal important links between oyster farming, environmental factors and husbandry strategies currently in practice in the surrounding tributaries of the Chesapeake Bay.
\(\)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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