Type of Document Dissertation Author Favret, Karen Perry Author's Email Address email@example.com, firstname.lastname@example.org URN etd-11062009-115056 Title Detection of Stress Biomarkers in Sperm, Embryonic, and Early Larval Stages of Aquatic Invertebrates Following Pesticide Exposure Degree Doctor of Philosophy (Ph.D.) Department Biological Sciences Advisory Committee
Advisor Name Title Lynn, John Committee Chair Galvez, Fernando Committee Member Whitehead, Andrew Committee Member Wilson, Vincent Committee Member Bargu-Ates, Sibel Dean's Representative Keywords
- flow cytometry
Date of Defense 2009-10-23 Availability unrestricted AbstractCurrent regulatory testing provides the basis for determining acceptable levels of pollutants in the environment, yet these acceptable levels of contaminants have resulted in undesirable consequences to organisms. The purpose of this dissertation was to test the hypothesis that biomarkers of cellular stress could be detected from sub-lethal exposure to pesticides in sperm and early life stages of broadcast spawning invertebrates. Exposures were conducted on oyster (Crassostrea virginica), mussel (Mytilus galloprovincialis and Dreissena polymorpha), and sea urchin (Lytechinus variegatus) embryos and larvae for 4 and 24 h to Bayluscide® and Roundup®. DNA fragmentation, a characteristic of apoptotic cells, was detected with the TUNEL method and an ELISA. Hsp70 expression was detected with Western blotting and quantified with densitometry. Sperm were exposed to the pesticides for 20 min and analyzed for cellular effects using flow cytometry.
Apoptosis levels often revealed a bell-shaped dose response in which there was a threshold concentration that elicited a change from apoptotic induction to necrosis. This change suggests irreversible damage to the organism has occurred and it is no longer using apoptosis as a defensive mechanism. The apoptotic results also revealed differential response levels among species despite very similar developmental stages. Hsp70 isoform expression was variable in controls and treatments of the majority of exposures. Therefore, it was concluded that this biomarker is unsuitable for use in early life stages of these species. Flow cytometric analyses of sperm viability biomarkers revealed that MitoTracker® was a reliable indicator for detecting changes in mitochondrial membrane polarization from Bayluscide® exposures, FITC-peanut agglutinin (PNA) reported acrosome reaction in two test species after Roundup exposures, and the SYBR®-14/propidium iodide (PI) assay only detected compromised membranes with Roundup® exposures as PI did not bind in the presence of Bayluscide®.
If the damage incurred at these stages does translate to lower fertilization success and abnormal development, then it is probable that reproductive competence will also be affected. Once long-term effects are established, detecting damage to sperm and early life stages can provide insight into the sub-lethal concentrations that may seemingly appear safe for an organism but can potentially pose serious risks to the population.
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