One noticeable service that microbiota provide for their hosts is protection from pathogens (Kamada et al., 2013). However, in compromised hosts or under (un)favorable 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 has a large impact on the population dynamics and evolution of affected organisms (Altizer et al., 2003), it is important to understand how the environmental factors and the resulting environmental stressors affect the composition and function of microbiota and the outcome of host–microbe interactions.
As suspension feeders, bivalves 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).
Widespread use of the herbicide atrazine has generated much research into its toxicity in aquatic systems. Atrazine, which has been banned from use by the European Union is nevertheless the 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. It is known to have high mobility through soil, so it can easily find its way into the aquatic environment (Marchini et al. 1988). Among host-bacterial interaction processes possibly disturbed by atrazine, the immune system is likely to be one of the more sensitive physiological systems (Fournier et al., 2000). The immune system contributes to host and host-symbiont homeostasis by eliminating foreign particles such as viruses, infectious bacteria or parasites (Fournier et al., 2000). 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 during spring and summer months (Agency for Toxic Substances and Disease Registry (2003) and paired with significant rainfall events, washes into the tributaries which carry it over oyster reefs and eventually into the Chesapeake bay.
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 is capable of altering gene expression across organisms. We believe atrazine may be inducing changes in bacterial composition in the eastern oyster that make it more susceptible to disease. For instance, atrazine exposure may prevent the colonization of beneficial "core" bacteria in eastern oyster lines.
In other documented aquatic ecosystems, the effects of atrazine have proven to be particularly pronounced for certain species. Indeed, 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 by running 16S sequencing in hatchery-reared spat will further our understanding of both oyster-prokaryote symbiosis and herbicide-prokaryote effects as well as increase our understanding of the effects herbicides have on a potential oyster core microbiome.
Methods
Oyster Acquisition and Stabilization
250 oyster (Crassostrea virginica) spat were purchased from Horn-Point Laboratory in April 2016. Once acquired, the oysters were allowed to stabilize in a holding tank for six months prior to treatment. The spat were placed in a large holding tank filled with approximately 300 L of pressure-treated water. The salinity of the tank was maintained at 25 parts per thousand using Instant Ocean sea salt. To minimize the buildup of ammonium and nitrate, water changes (25%) were conducted twice per week. In addition to frequent water changes, Kordon AmQuel Plus Ammonia Detoxifier/Conditioner and TLC Saltwater aquarium conditioner were used to remove Nitrate, Nitrite and Ammonia as needed.
Assigning Experimental Groups
Oysters were randomly separated into five groups of 50 oysters each. To prevent overcrowding of the organisms, each of these groups was then divided into a sub-group of 10 oysters. 3.0 mm square mesh sieves were used to hold each sub-group. No oyster was smaller than 5.0 mm long x 4.0 mm wide when placed in the mesh sieves.
Treatments
Each group of 50 oysters was assigned to one of five treatments: 0 μg/L Atrazine, 3 μg/L Atrazine, 10 μg/L Atrazine, 30 μg/L Atrazine, or 30 μg/L acetone.
The stabilization tank served as the holding chamber for the no-treatment control group (0 μg/L of atrazine, 0 μg/L acetone). Treated groups were removed from the holding tank and placed in separate glass tanks each containing 4 liters of water (salinty= 25 ppt). Atrazine was added to each tank according to treatment concentration. To mimic the heavy rainfall pattern surrounding the Chesapeake Bay, treated groups spent a total of 3 hours submerged in 2 liters of treated water within each glass tank twice per week. Each group was rinsed with 25 ppt water before being placed back into the holding chamber.
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.
4) The tags were compared with the reference database (Gold database, http://drive5.com/uchime/uchime_download.html) using UCHIME algorithm (UCHIME Algorithm. (http://www.drive5.com/usearch/manual/uchime_algo.html) to detect chimeric sequences, and then the chimeric sequences were removed. The data output of the above steps is shown in Table 1.
High quality reads were obtained from experimental groups “Orange 30” ________________, “Pink 10” _______________, “Green 3”_________________ Acetone 30, and ________________ from the control group. The general microbiome of the Crassotrea virginica tissue samples was assembled in a total of __________ base pair reads. The general microbiome revealed bacteria thought to be involved in pathways related to immunity, and normal cell functioning.
Results