Introduction
The largest estuary in the United States, the Chesapeake, once teemed with oysters. In fact, its name comes from the Algonquin word Chesepioooc, which translates to the Great Shellfish Bay (3). The National Oceanic and Atmospheric Administration (NOAA) now reports that the native oyster population of the Chesapeake Bay has diminished to 1% the original value (6). Today we understand that the microbiota of marine mollusks are vitally important for their survival, homeostasis, and development (McFall-Ngai et al., 2013). Widespread use of the herbicide atrazine has generated much research into its toxicity in aquatic systems, in this study we investigate how ecologically relevant concentrations of atrazine affect the microorganism-host interactions of the eastern oyster .
Atrazine, which has been banned from use by the European Union is nevertheless the second most widely used herbicide in the United States, with an estimated annual production of 76 million pounds. When applied, atrazine acts as a chemical contaminant of both surface and ground waters. Atrazine (6-Chloro-n-ethyl-n’-(1-methylethyl)-triazine-2,4-diamine) is a synthetic herbicide commonly used on crops like corn, sugar cane, and evergreens, especially during spring and summer months (Agency for Toxic Substances and Disease Registry, 2003) and paired with significant rainfall events, washes directly into tributaries which carry it over oyster reefs for extended periods of time. One of the beneficial roles microbiota have in oysters is to protect against pathogens and environmental stressors, however, the response of resident bacteria in the host to chemical contamination remains largely unexplored.
The Eastern oyster, Crassostrea virginica, is one of the most frequently cultivated bivalve species in the world and is typically reared in estuarine environments that have become increasingly threatened by exposure to pollutants. Among pollutants, herbicide/pesticide contamination of shellfish has become more common in estuarine areas over the past several decades due, in part, to chemical run-off from terrestrial agriculture (Banerjee et al. 1996, khan et al. 2017). The microbial communities of suspension-feeding bivalves' include both resident and transient microbiota. Bivalves have the ability to filter large quantities of water (e.g., 3–9 L/h/g dry mass for oysters; Newell et al. 2005, Cranford et al. 2011), and so come into contact with, and pass through their bodies, both free-living and particle-associated microbes. The bacteria with which they interact and harbor have the potential to be particularly important to physiological and biochemical enantiostasis.
Agricultural pollutants may have major ecological consequences and could endanger organism growth, reproduction or survival by altering the beneficially associated microbiota (Banerjee et al., 1996). In this study, one 16s rRNA gene amplicon sequencing event was used to characterize the microbiome of hatchery raised C. virginica juveniles. We sought to characterize how atrazine effects the bacterial composition of juvenile oysters after having been exposed to environmentally relevant concentrations of atrazine three times a week for a period of one-month. Extending the analysis to resident microbial communities offers a unique opportunity to understand how host and resident bacteria altogether respond to chemical contaminations. Countless xenobiotic compounds, including pesticides, pharmaceuticals, and personal care products, among others, are continuously introduced into the environment and have been detected at concentrations up to μg/L levels in surface waters. The presence of herbicides in aquatic environments is one of the major challenges for the preservation of a bivalves essential microbial environment. One noticeable service that microbiota provide for their hosts is protection from pathogens (Kamada et al., 2013). Indeed, in compromised hosts or under unfavorable environmental conditions, the symbionts themselves have been understood to act as opportunistic pathogens (Garnier et al., 2007; Cerf; Bensussan and Gaboriau-Routhiau, 2010; Olson et al., 2014). As disease prevelance has a large impact on the population dynamics and evolution of affected organisms (Altizer et al., 2003), it is important to understand how environmental factors and their resulting environmental stressors affect the composition and function of microbiota and the outcome of host–microbe interactions in C. Virginica.
As suspension feeders, oysters interact significantly with living and non-living particles in the seston, including bacteria, as they filter large quantities of water per unit time. It is thus unsurprising that they harbor an order of magnitude more bacteria than does the water in which they live (Colwell & Liston, 1960; Cavallo et al., 2009). Next-generation sequencing, although by no means free of biases (Fierer and Lennon, 2011; Sergeant et al., 2012; Cai et al., 2013) enables detailed characterization of microbial community composition and dynamics, including rare phylotypes (Huse et al., 2008) that can act as a seed bank and mediate community response to environmental change (Caporaso et al., 2012; Pedros-Alio, 2012; Sjostedt et al., 2012).
The U.S. Safe Drinking Water Act established the maximum contaminant level of Atrazine to be 3 µg/L (EPA). However, a study conducted by the USDA in 2006 found the concentration of Atrazine in the Chesapeake Bay watershed to be 30 ug/L, 10x the maximum contaminant safety level (USDA, 2006). While advertised as safe, independent studies have shown atrazine to cause chemical castration in frogs (Hayes et. al., 2010), and increased menstrual cycle irregularity in humans (Cragin et. al., 2011). Results like these suggest that atrazine may also be inducing changes in the bacterial composition residing within the eastern oyster, an organism which regularly interacts with the substance in its ecosystem. This constant exposure to pollution may make the keystone species more susceptible to disease (Cragin et. al., 2011). Indeed, in other documented aquatic ecosystems, the effects of atrazine have proven to be particularly pronounced. It has been documented that exposures to concentrations as low as 0.1 parts per billion of atrazine in surface water have adversely affected frogs in causing the male gonads to produce eggs – effectually turning males into hermaphrodites (DeLorenzo et al. 2001; Lynn 2017). The effects of atrazine at more environmentally realistic concentrations are far less clear, and the potential uninterrupted and adjuvant effects resulting from use of atrazine on the survival and growth of Crassostrea virginica are simply not known.
The Chesapeake Bay has witnessed staggering losses to oyster populations over the past century, reported to be down by 97% when likened to early records (Chesapeake Bay Foundation 2016). Atrazine is commonly used in and around agricultural fields in the Chesapeake Bay watershed (USDA). For this reason, it was chosen to be the focus in this study examining the effects of herbicide-induced bacterial composition changes. The objective of the present study was to analyze the microbiota of juvenile oyster specimens in order to test what the response of resident microbial communities is to environmentally relevant concentrations of atrazine. Here, we aim to assess the impact of long-term exposure to pollutants on microbial communities, as well as to evaluate the potential impact of changes in microbial communities on host xenobiotic evolution and susceptibility to environmental chemicals, two crucial but still unresolved questions in ecotoxicology.
Methods
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 using that concentration in the experiement: