Tracey B. Schock

NMR-based microbial metabolomics and the temperature-dependent coral pathogen Vibrio coralliilyticus.

Coral bleaching occurs when the symbioses between coral animals and their zooxanthellae is disrupted, either as part of a natural cycle or as the result of unusual events. The bacterium Vibrio coralliilyticus (type strain ATCC BAA-450) has been linked to coral disease globally (for example in the Mediterranean, Red Sea, Indian Ocean, and Great Barrier Reef) and like many other Vibrio species exhibits a temperature-dependent pathogenicity. The temperature-dependence of V. corallillyticus in regard to its metabolome was investigated. Nuclear magnetic resonance (NMR) spectra were obtained of methanol-water extracts of intracellula rmetabolites (endometabolome) from multiple samples of the bacteria cultured into late stationary phase at 27 degrees C (virulent form) and 24 degrees C (avirulent form). The spectra were subjected to principal components analysis (PCA), and significant temperature-based separations in PC1, PC2, and PC3 dimensions were observed. Betaine, succinate, and glutamate were identified as metabolites that caused the greatest temperature-based separations in the PC scores plots. With increasing temperature, betaine was shown to be down regulated, while succinate and glutamate were up regulated.

Evaluation of Pacific White Shrimp (Litopenaeus vannamei) Health during a Superintensive Aquaculture Growout Using NMR-Based Metabolomics

Success of the shrimp aquaculture industry requires technological advances that increase production and environmental sustainability. Indoor, superintensive, aquaculture systems are being developed that permit year-round production of farmed shrimp at high densities. These systems are intended to overcome problems of disease susceptibility and of water quality issues from waste products, by operating as essentially closed systems that promote beneficial microbial communities (biofloc). The resulting biofloc can assimilate and detoxify wastes, may provide nutrition for the farmed organisms resulting in improved growth, and may aid in reducing disease initiated from external sources. Nuclear magnetic resonance (NMR)-based metabolomic techniques were used to assess shrimp health during a full growout cycle from the nursery phase through harvest in a minimal-exchange, superintensive, biofloc system. Aberrant shrimp metabolomes were detected from a spike in total ammonia nitrogen in the nursery, from a reduced feeding period that was a consequence of surface scum build-up in the raceway, and from the stocking transition from the nursery to the growout raceway. The biochemical changes in the shrimp that were induced by the stressors were essential for survival and included nitrogen detoxification and energy conservation mechanisms. Inosine and trehalose may be general biomarkers of stress in Litopenaeus vannamei. This study demonstrates one aspect of the practicality of using NMR-based metabolomics to enhance the aquaculture industry by providing physiological insight into common environmental stresses that may limit growth or better explain reduced survival and production.

Application of NMR-based metabolomics for environmental assessment in the Great Lakes using zebra mussel (Dreissena polymorpha)

Abstract Zebra mussel, Dreissena polymorpha, in the Great Lakes is being monitored as a bio-indicator organism for environmental health effects by the National Oceanic and Atmospheric Administration’s Mussel Watch program. In order to monitor the environmental effects of industrial pollution on the ecosystem, invasive zebra mussels were collected from four stations—three inner harbor sites (LMMB4, LMMB1, and LMMB) in Milwaukee Estuary, and one reference site (LMMB5) in Lake Michigan, Wisconsin. Nuclear magnetic resonance (NMR)-based metabolomics was used to evaluate the metabolic profiles of the mussels from these four sites. The objective was to observe whether there were differences in metabolite profiles between impacted sites and the reference site; and if there were metabolic profile differences among the impacted sites. Principal component analyses indicated there was no significant difference between two impacted sites: north Milwaukee harbor (LMMB and LMMB4) and the LMMB5 reference site. However, significant metabolic differences were observed between the impacted site on the south Milwaukee harbor (LMMB1) and the LMMB5 reference site, a finding that correlates with preliminary sediment toxicity results. A total of 26 altered metabolites (including two unidentified peaks) were successfully identified in a comparison of zebra mussels from the LMMB1 site and LMMB5 reference site. The application of both uni- and multivariate analysis not only confirmed the variability of altered metabolites but also ensured that these metabolites were identified via unbiased analysis. This study has demonstrated the feasibility of the NMR-based metabolomics approach to assess whole-body metabolomics of zebra mussels to study the physiological impact of toxicant exposure at field sites.

Trace element biodistribution in the American alligator (Alligator mississippiensis)

Routine monitoring of contaminant levels in wildlife is important for understanding chemical exposure and ultimately the link to ecosystem and human health. This is particularly important when the monitored species is recreationally hunted for human consumption. In the southeastern United States, recreational alligator harvesting takes place annually and in locations that are known to be contaminated with environmental pollutants. In this study, we investigated the biodistribution of trace elements in the American alligator (Alligator mississippiensis) from five sites in Florida, USA. These sites are locations where annual recreational alligator harvesting is permitted and two of the sites are identified as having high mercury contamination with human consumption advisories in effect. We utilized routinely collected monitoring samples (blood and scute), a commonly consumed tissue (muscle), and a classically analyzed tissue for environmental contaminants (liver) to demonstrate how the trace elements were distributed within the American alligator. We describe elemental tissue compartmentalization in an apex predator and investigate if noninvasive samples (blood and scute) can be used to estimate muscle tissue concentrations for a subset of elements measured. We found significant correlations for Hg, Rb, Se, Zn and Pb between noninvasive samples and consumed tissue and also found that Hg was the only trace metal of concern for this population of alligators. This study fills a gap in trace elemental analysis for reptilian apex predators in contaminated environments. Additionally, comprehensive elemental analysis of routinely collected samples can inform biomonitoring efforts and consumption advisories.

Metabotyping of a Protected Non-Model Organism, Green Sea Turtle (Chelonia mydas), using 1 H NMR Spectroscopy and Optimized Plasma Methods for Metabolomics

The metabolomic fingerprints of a protected sea turtle species have been investigated for the first time using a nuclear magnetic resonance (NMR)-based metabolomics approach. We emphasized method development of optimal plasma filtration conditions (filter type, washing techniques, extract stability) for green turtles and other organisms, while also using the National Institute of Standards and Technology (NIST) Standard Reference Material 1950 (SRM 1950) Me- tabolites in Human Plasma for quality control. We surveyed the blood plasma metabolomic fingerprints of Hawaiian green sea turtles representing a wide range of physiological conditions that include varying disease states, behavioral con- ditions, and locales. The turtles sampled were free-swimming (n=5 from Hualalai on the west coast of the island of Ha- waii), basking (n=7 from Hualalai), free-swimming tumor-free (n=3 from Kapoho, east coast of Hawaii), or free- swimming afflicted with external tumors (n=5 from Kapoho) caused by the disease, fibropapillomatosis (FP). The me- tabolomic profiles and the specific metabolites that differed among individual turtles are discussed. This optimized tool and the annotated metabolic profiles will benefit future investigations into the behavioral and disease conditions of the green turtle.

Examining heat treatment for stabilization of the lipidome

AIM To confidently determine lipid-based biomarkers, it is important to minimize variation introduced during preanalytical steps. We evaluated reducing variation associated with lipid measurements in invertebrate sentinel species using a state-of-the-art heat treatment technique. MATERIALS AND METHODS Earthworms (Eisenia fetida), house crickets (Acheta domestica) and ghost shrimp (Palaemonetes paludosus) were euthanized either by flash freezing or heat treatment. For both experiments, samples were either immediately extracted after removal from -80°C storage or incubated on ice for one hour prior to sample weighing and extraction. Lipidomics was performed on resulting extracts using liquid chromatography high resolution tandem mass spectrometry. LipidMatch and LipidSearch were used for lipid identification. RESULTS Lipid enzymatic products (e.g., phosphatidylmethanols, diglycerides, lysoglycerophospholipids and ether-linked/oxidized lysoglycerophospholipids), were in higher concentrations in flash-frozen samples, when compared with heat-treated samples. Results suggest that heat treatment reduces phospholipase A and phospholipase D activity. CONCLUSION Heat treatment reduced enzymatic products and increased precursors of these enzymatic products. We believe heat treatment warrants a closer interrogation for improving the robustness of lipid biomarker research, especially in tissue samples, where enzyme stabilizers are difficult to apply, and for use in field studies, where the stabilization of the collected sample is critical.

Effects of heat-treatment on the stability and composition of metabolomic extracts from the earthworm Eisenia fetida

Environmental metabolomics studies employing earthworms as sentinels for soil contamination are numerous, but the instability of the metabolite extracts from these organisms has been minimally addressed. This study evaluated the efficacy of adding a heat-treatment step in two commonly used extraction protocols (Bligh and Dyer and D2O phosphate buffer) as a pre-analytical stabilization method. The resulting metabolic profiles of Eisenia fetida were assessed using principal component analysis and NMR spectral evaluations. The heated Bligh and Dyer extractions produced stabilized profiles with minimal variation of the extracted metabolomic profiles over time, providing a more suitable method for metabolomic analysis of earthworm extracts.

Evaluating mercury concentrations and body condition in American alligators (Alligator mississippiensis) at Merritt Island National Wildlife Refuge (MINWR), Florida

Concentrations of mercury (Hg) are not well studied in free-ranging wildlife. Atmospheric deposition patterns of Hg have been studied in detail and have been modeled for both global and specific locations and often correlate to environmental impact. However, monitoring the impact of Hg deposition in wildlife is complicated due to local environmental conditions that can affect the transformation of atmospheric Hg to the biologically available forms (e.g., rainfall, humidity, pH, the ability of the environment to methylate Hg), as well as affect the accessibility to organisms for sampling. In this study, Hg concentrations in blood samples from a population of American alligators (Alligator mississippiensis) at Merritt Island National Wildlife Refuge (MINWR), FL, USA, over a seven-year period (2007 to 2014; n=174 individuals) were examined to assess Hg variation in the population, as well as the difference in Hg concentration as a function of health status. While most of this population is healthy, 18 individuals with low body mass indices (BMI, defined in this study) were captured throughout the sampling period. These alligators exhibited significantly elevated Hg concentrations compared to their age/sex/season matched counterparts with normal BMI, suggesting that health status should be taken into account when examining Hg concentrations and effects. Alligator blood Hg concentrations were related to the interaction of age/size, sex, and season. This study illustrates the value of a routinely monitored population of large predators in a unique coastal wetland ecosystem, and illuminates the value of long-term environmental exposure assessment.