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The Aquatic Research Consortium: Genomic, Proteomic and Organismal Responses of Aquatic Organisms to Hypoxia and Toxic Metals
FY 2006 Abstract (Year 4)
Many estuaries are adversely affected by chemical contamination and nutrient-overload due to rapidly increasing human coastal populations, urbanization, industrial effluents, and agricultural run-off. The detrimental effects of increased levels of environmental contaminants such as toxic metals upon aquatic ecosystems have been well-documented. In addition, hypoxia-related mass mortalities and population declines in aquatic organisms are often a result of elevated levels of estuarine nutrients derived from anthropogenic sources. Therefore, studies elucidating the response of estuarine organisms to both hypoxia and metals are important in ecological risk assessment. Furthermore, it is expected that important ìbiomarkerî responses will be determined, allowing for more accurate and proactive decisions to be made regarding resource management.
The proposed research will provide much needed information by investigating the effects of hypoxia, cadmium and chromium, hypoxia/cadmium, and hypoxia/ chromium combined exposures in fish at the molecular and organismal levels. This proposal requests the fourth phase of funding for the Aquatic Research Consortium (ARC). The proposed research will entail a collaborative and integrative effort between scientists at the Gulf Coast Research Laboratory (GCRL) of The University of Southern Mississippi and Texas State University (TSU).
The first three phases of this program focused on the establishment of genomic and proteomic tools for investigation of stress responses, with a focus on hypoxia and polycyclic aromatic hydrocarbons, in two environmentally relevant estuarine species (the grass shrimp, Palaemonetes pugio, and the sheepshead minnow, Cyprinodon variegates) and two well-characterized small fish models (Japanese medaka, Oryzias latipes, and swordtails, Xiphophorus spp). The fourth phase of this program will continue to build upon information gained during the previous phases regarding the hypoxic response of these four species.
Our studies with grass shrimp indicate that expression of mitochondrial genes is coordinately regulated in response to hypoxia. We will extend these studies in more detail in both grass shrimp and sheepshead minnows by measuring hypoxia-induced mitochondrial mRNA and protein levels. mRNA will be measured using real-time qPCR. Protein expression will be measured using a ProteomeLab PFD system to separate proteins in two-dimensions, followed by characterization of differentially expressed proteins using MALDI-TOF mass spectrometry. We expect that our proposed studies will show that enhanced expression of mitochondrial genes will be due to amplification of mitochondrial genome relative to the nuclear genome. In that case, measurement of mitochondrial protein and/or DNA may provide us with a simple biomarker of hypoxia exposure. Studies on wild-caught animals from hypoxic and normoxic locations will test the validity of the biomarker in the field. Exposure to cadmium and chromium is associated with a variety of adverse health effects, including cancer. Identification of genes and proteins whose expression is altered by chromium and cadmium exposure may increase our understanding of their mechanism of action and may lead to discovery of specific biomarkers of exposure. Since the two metals and hypoxia (chronic and intermittent) are known to induce oxidative stress, it appears likely that combined exposure to metals and hypoxia will result in injury extending well beyond that caused by individual exposures. To our knowledge, such co-exposures have not been conducted to date. Hence we propose to perform co-exposure experiments, using sensitive early life stages of our aquatic animal models (Cyprinodon variegatus and Oryzias latipes). For these studies, hypoxia, metal, and hypoxia/metal-induced changes in gene and protein expression, physiology, and morphology will be assessed in larval fish. Thus, the proposed research will attempt to characterize species- and life stage-specific responses of fish to natural and anthropogenic stressors at the molecular, physiological, and organism levels. This information will be integrated with results from the previous ARC phases (including reproductive data) to estimate possible higher-level (i.e., population and ecosystem) effects of exposure to common environmental stressors.