Stress response of bivalves: impact of copper on filtration rate and microbial communities of oysters (Crassostrea virginica) Hannah Ryon Northwestern University Evanston, IL 60201 Advisor: Anne Giblin The Ecosystems Center Marine Biological Laboratory Woods Hole, MA 02543 Semester in Environmental Science 2016 Abstract Copper can have detrimental effects for organisms in aquatic environments, especially with increasing modern inputs from industrial waste, sewage, paint, and pressure treated lumber. Although there have been many studies on the lethal implications, there is not much known about the sub-lethal effects of copper on organisms. This study looks at the impact of copper exposure on filtration rate and microbial communities of eastern oysters. To test these two parameters, I setup tanks with varying concentrations of dissolved copper and a total of 20 oysters from Little Pond, in Falmouth, MA. I then measured the change in phytoplankton in the water over time to determine filtration rate. I also extracted and sequenced the DNA in the feces, pseudofeces, and on the shell. Finally, I determined the metal content of both the body and the gut of the exposed oysters. In this study, I found that the filtration rate of the oysters decreased with increasing concentrations of copper. However, the filtration rate rapidly recovers after being removed from tanks with copper. In the microbial component of this study, I determined that eastern oysters do not have a resident gut microbial community but they do have a resident shell community. This shell community is highly diverse, and it has denitrifying genera present. In the water and on the shell the microbial diversity decreases with exposure to copper. Finally, I determined that the oysters increase the amount of copper absorbed with increasing exposure. However, at around 600ppm the oysters become saturated with copper. Additionally, the gut contained higher concentrations of copper than the body of the oysters because the gut is used to expel excess copper. This data suggests that copper can have many sub-lethal effects on the eastern oyster at concentrations below the toxic concentration. Introduction All organisms require trace amounts of copper in order to survive. Copper is a metal that can be naturally found in trace amounts in animals, soils, plants and water (Diseases and Contaminants). In aquatic systems, copper is a relatively common metal ion. However, when in excess it becomes one of the most toxic ions in aquatic systems (Solomon 2009). This is why it is so important to understand the interactions of organisms with copper in aquatic environments. Typically, natural background levels of copper enter water bodies through weathering and material blown in by the wind. However, in present day, copper concentrations in aquatic environments can be greatly increased due to inputs from air pollution, industrial waste, and sewage (Bryan 1971). In marine environments two other major sources of copper are boat bottom paint and pressure treated lumber (Schiff 2004 & Stook 2005). This increase in dissolved copper can have significant ramifications for the organisms in that environment. One way in which excess copper can be detrimental to organisms is by disrupting cellular processes. When copper is abundant in an organism it then displaces other ions by binding to metal-binding sites or other nonspecific sites in things like enzymes or DNA. This is highly disruptive to metabolic processes and can lead to the eventual death of the organism (Goldstein 1986). Additionally, excess copper in an organism can catalyze reactions, which produce oxyradicals. This is extremely dangerous to an organism because oxyradicals can cause their nervous system to shut down (Goldstein 1986). The gills of aquatic organisms can also be greatly damaged by excess copper in the body because the efficiency of respiratory mitochondria in the gills falls with increased copper concentration in oysters (Collins 2010). In this study I will be focusing on the impact of copper on eastern oysters (Crassotrea virginica). Oysters are suspension feeders, meaning they filter phytoplankton and other nutrient particles out of the water. The suspended particles that they siphon in are eventually separated into feces and pseudofeces or incorporated into the organism. The pseudofeces is the material that the oyster determines is not nutritious and it is rejected prior to digestion. The feces is excreted on the opposite end of the oyster after it is digested and the necessary material is extracted. These oysters pay a large role in maintaining ecosystems, as their consumption patterns allow nitrogen to cycle and they help to prevent eutrophication (Songsangjinda 1998). It has also been suggested that the microbes that they harbor assist in denitrification (Kellogg 2014). Another reason for the importance of the oysters in Cape Cod is because they are a large component of the shellfish industry, which boosts the economy and benefits tourism. As copper amounts increase in the environment, it is vital that we understand the implications of this on the Cape Cod oyster population (Diseases and Contaminants 2015). Many studies have shown the lethal effects of copper on organisms, however, little is known about the sub-lethal impact of copper on organisms. Previous studies have shown that reproduction can be severely hindered by exposure to increased copper concentrations (Hornberger 2000). This shows that organisms can have sub- lethal changes due to copper, and this experiment will aim to determine sub-lethal effects in a short-term setting. I will be using filtration rate as a test of these sub lethal effects on the organism, by showing the impact of copper on phytoplankton consumption. Additionally, I plan to examine the impact of copper on the residential microbial communities within the organism. Little is known about the species in these communities, or if the community is present at all, but any change in its composition could be an indicator of stress on the oyster. Additionally, it is important to understand the impact of copper on these communities, as they are involved in some vital processes in the environment such as denitrification. Finally, I will examine how the metal is incorporated into the organism and which parts of the body are most contaminated. This will determine if metal is actually absorbed by the organism, or if it just passes through its system. These tests can also point to the mechanism of storing or expelling the copper from the system. Methods Lab Setup This experiment was conducted entirely in a lab setting. I set up 5 different tanks with four oysters of similar size in each, as shown in Figure 1. The oysters were collected from the Little Pond oyster farm in Falmouth, MA. Each tank is filled with seawater filtered through a 20micron filter and a 1micron filter, in order to remove any phytoplankton present in the inflowing seawater. The water was also bubbled with air throughout the course of the entire experiment, to ensure uniform distribution of copper and phytoplankton. In each tank there was a different concentration of copper dissolved in the water: 0ppm, 0.05ppm, 0.1ppm, 0.2ppm, and 0.3ppm. The copper was added using a Cu (II) 1000ppm stock solution. The oysters were allowed to sit in the filtered seawater without any phytoplankton inputs or copper additions for two days. This was done in order to flush their systems of any prior food or other contaminants from their previous environments. I also predict that this will eliminate any microbiomes present due to anything they consumed prior to being moved to the lab. During test periods, the tanks were cleaned each morning and the water was replaced. Copper and algae was again added to the tanks after cleaning. The algae was obtained from the Marine Resources Center at the Marine Biological Laboratory of Woods Hole, and it was RotiGrow Plus Omega Algal Blend. Filtration Rate The first objective of this experiment was to determine the impact of copper on the filtration rate of the organisms. In order to determine filtration rate in each tank, I measured the change in chlorophyll a concentration over time. As the algae is filtered out of the water column, the amount of chlorophyll a in the water will decrease. I measured chlorophyll a in the water by taking a subsample of the water from each tank and using a Turner Designs 10A Flourometer. I was then able to determine filtration rate by utilizing the following equation: 푉1−푉2 Filtration Rate = 푡 Where V is the concentration of chlorophyll a measured in ug/L, and t is time in hours. Filtration rate was measured over 4 different trials of 1 day experiments, with fresh filtered sea water, copper, and food supply each day. There were typically approximately 2 days between each trial. During the second and third trials the oysters were placed on platforms for feces and pseudofeces collection, as shown in Figure 2. On the second day of test one of the oysters in the 0.2ppm tank died. The organisms were then starved without copper for two days and the filtration rate was measured to the organisms’ ability to recover. Microbial Communities In order to determine the diversity of microbial communities present before and after copper exposure, I focused on microbes in the feces, pseudofeces, and on the shell of the oysters. During the third day of filtration tests, in the 0ppm and 0.3ppm tanks, I collected a subsample of feces and pseudofeces from the collection tin of three of the oysters. Only three oysters in each of the tanks were sampled due to limitations on costs. Additionally, I scraped the surface of the shells of these three oysters and collected any material present. After collecting the feces, pseudofeces, and shell matter from three oysters in both 0ppm and 0.3ppm, I then completed DNA extractions for each of these.