As a result of the Deepwater Horizon oil spill, millions of gallons of oil have leaked into the Gulf of Mexico and much of that oil has and will continue to wash up along the Gulf Coast of the United States. In an attempt to minimize the amount of oil to reach the coast, an unprecedented of amount of dispersant has been used both on surface oil and at the source of the leak. The impact of this unprecedented use of dispersant on the marine environment, food webs and the bioavailability of oil remains to be investigated. Furthermore, the microbial degradation of oil and dispersed oil in the marine environment or estuarine marshes can produce very low oxygen levels (hypoxia) that will further stress marine and estuarine invertebrates. Unfortunately, very little information is available on the impact of oil or dispersed oil and additional abiotic stressors (hypoxia) on the physiology of marine and estuarine invertebrates. Nematostella vectensis is a sea anemone (an organism related to reef-building corals) found in salt marshes along the Gulf of Mexico and Atlantic coast of the United States. This project will use Nematostella as a model to address the urgent need to understand the physiological responses of estuarine invertebrates to oil exposure, combined exposure of oil and dispersant and possible synergism between oil exposure and hypoxia (low oxygen). First, Nematostella will be collected from Gulf Coast populations. Contaminant load, energetic stores and reproductive status will be quantified. Molecular techniques will be used to determine which genes are affected ("turned on" or "turned off") in anemones from oil-exposed sites. Second, laboratory experiments will be conducted to determine the effects of oil exposure and combined exposure to oil and dispersant under normal oxygen (normoxic) and hypoxic conditions. Brine shrimp will be reared in the presence of a range of concentrations of oil, dispersant, and oil with dispersant. These shrimp will be fed to Nematostella under normoxic and hypoxic conditions. Effects of exposure will be characterized by assessing changes in gene expression, lipid analysis, histological examination and biochemical assays. The results of these experiments will provide insight into the different molecular and cellular processes that are used to protect the organism from combinations of stressors that are associated with the oil spill and exposure to oil or dispersed oil. This project will also enable development of biomarkers that can be used to assess responses of organisms collected in the field.
Broader Impacts: This project will provide insight into the ecological consequences of the Deepwater Horizon oil spill. Results from field sampling will be posted and linked with other emerging results (e.g., EPA sediment analysis). Gene expression and sequence data will be curated, and posted to publicly accessible databases. Two PhD students will be trained in lipid analysis and ecological genomics. This project will identify cellular and molecular responses of a cnidarian (sea anemone, related to reef-building corals) to oil exposure through an understudied route of exposure (feeding). In addition this project will provide direct measurements of gene expression, lipid stores and contaminant burdens an indicator species of anemones from potentially impacted populations along the Gulf Coast. The identification and characterization of various pathways being affected by oil exposure and related stressors (e.g., hypoxia) will assist in development of ecological forecasting tools to predict the physiological responses of organisms and subsequent impacts on local populations and ecosystems.
As a result of the Deepwater Horizon oil spill, salt marsh organisms are being exposed to fresh, weathered and dispersed oil, oil-based compounds and tarballs. Virtually nothing is known regarding the sublethal physiological responses of estuarine invertebrates to petroleum-based pollutants, despite the importance of these animals to estuarine food webs and widespread use of benthic diversity as an indicator of ecosystem health. Nematostella vectensis (starlet sea anemone) is an estuarine denizen found along the Gulf of Mexico and eastern Atlantic seaboard of the United States. This project will use Nematostella as a model to address the urgent need to understand the physiological responses of estuarine invertebrates to oil exposure, combined exposure of oil and dispersant and possible synergism between oil exposure and UV exposure resulting in increase photo-toxicity. The classic hydrocarbon stress response in vertebrates is a result of activation of the aryl hydrocarbon receptor (AHR) protein by binding of a hydrocarbon to the ligand domain resulting in translocation of the receptor to the nucleus where is interacts with ARNT (AHR nuclear translocator protein) to form a transcriptional complex that up-regulates a battery of Phase I and Phase II enzymes that are responsible for detoxifying hydrocarbons by altering their chemical structure to increase their water solubility and aid in excretion. The induction of these enzymes, particularly the Phase I enzyme CYP1A, is a classic molecular biomarker of hydrocarbon exposure used in vertebrate models. However, all published evidence to date suggests that the AHR does not regulate the hydrocarbon stress response in invertebrates. We choose eight 24-hour treatments (Seawater, SW with dispersant, SW with oil, and SW with oil & dispersant; repeated these treatments to include a 6 hour UV exposure to simulate mid-day exposure) with three biological replicates with each replicate consisting of six Nematostella. Three of the Nematostella from one replicate were removed and pooled for RNA isolation to be used in the extensive investigation of gene expression profiling by RNA-seq, a technique that use high-throughput DNA sequencing to determine changes in gene expression. The RNA-seq was performed by generating an average of ~100 million sequences per sample and comparing each sequence read to a database of known Nematostella genes. The remaining three Nematostella per replicate were pooled and flash frozen in liquid nitrogen and cryopreserved for simultaneous analysis of oil residues and lipids. Changes in gene expression will be correlated with concentrations of oil residues detected in Nematostella tissues. Lipid analysis will provide insight into the effects of the oil stress on the physiological status of the animals and can yield potential information about oxidative stress. Interestingly, oil, dispersant, and dispersed oil exposures caused very minor change in gene expression in all three treatments with little overlap between the different treatments. This data suggest a very weak toxic response to the oil. However when you couple oil and UV exposure, the photo-oxidation of the oil resulted in a drastic increase in the toxic response in Nematostella. To briefly summarize the results, oil exposure alone resulted in differential expression of 38 genes (out of ~24,000), whereas exposure to oil and UV resulted in the differential regulation of 1,268 genes, of which 837 were specific to photo-oxidized oil and the remaining 431 were related to UV exposure alone. A perusal of the top 20 genes upregulated by photo-oxidized oil shows a significant number of Phase I and Phase II enzymes, as well as genes involved in the oxidative stress response. However, none of the Phase I or Phase II enzymes upregulated in Nematostella correspond to known AHR genes typically induced in vertebrate models. Thus, we have identified a novel set of Phase I and Phase II enzymes that may represent a unique hydrocarbon detoxification and metabolism mechanism in invertebrate organisms. Additional research into these mechanisms will provide insight into how marine invertebrates respond to hydrocarbon toxicity and will allow us to make predictions about the effects of future oil spills on the health of marine organisms.
