Aquatic hypoxia, or low dissolved oxygen, is a growing environmental concern and has detrimental effects on many fishes. Research on fish responses to hypoxia includes whole animal studies as well as organ culture systems. In this thesis, the gulf killifish, Fundulus grandis, was used to determine the interaction between hypoxic exposure and four common euthanasia techniques on selected blood and gill variables and to develop an in vitro gill incubation system. Euthanasia techniques had differential effects on blood and gill, with the common fish anesthetic MS-222 having the greatest effects, but none altered the response to hypoxia. During the development of gill culture, the highest gill viability was measured in isosmotic seawater medium and shorter incubation times. This study provides insight into the applicability of different euthanasia techniques in hypoxia research, as well as preliminary observations on an incubation system for isolated gills.

This research examined the effects of acclimation to lowered salinity, elevated temperature, and hypoxia on aerobic metabolism of the Gulf killifish, Fundulus grandis, a common estuarine resident of the Gulf of Mexico. Standard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS), and critical oxygen tension (Pcrit) were each influenced by one or more acclimation treatments. Assessing the consistency of these traits measured in the same individuals over time, all were found to be significantly repeatable with no indication that the repeatability of any traits was affected by acclimation conditions. Significant correlations were found between SMR and Pcrit (positively correlated), between SMR and AAS (negatively correlated), between MMR and AAS (positive), and between AAS and Pcrit (negative). This study, therefore, documents the effects of acclimation on these traits, their repeatability, and correlations among them. It further suggests that repeatability of these traits is not context dependent.

Fundulus is a diverse and widespread genus of small teleost fish of North America. Due to its high tolerance for physiochemical variation (e.g. temperature, oxygen, salinity), Fundulus is a model organism to study physiological and molecular adaptations to environmental stress. The thesis focuses on patterns of protein expression in Fundulus heteroclitus and F. grandis.The patterns of protein expression were investigated using traditional methods of enzyme activity measurements and recent proteomic approaches. The findings of the study can be used to guide future studies on the proteomic responses of vertebrates to environmental stress. Chapter 2 focuses on measurement of the temporal effects of oxygen treatments on the maximal specific activities of nine glycolytic enzymes in liver and skeletal muscle during chronic exposure (28d) of Fundulus heteroclitus. The fish was exposed to four different oxygen treatments: hyperoxia, normoxia, moderate hypoxia, and severe hypoxia. The time course of changes in maximal glycolytic enzyme specific activities was assessed at 0, 8, 14 and 28 d. The results demonstrate that chronic hypoxia alters the capacity for carbohydrate metabolism in F. heteroclitus, with the important observation that the responses are both tissue- and enzyme-specific. Chapter 3 studies the effect of tissue storage on protein profile of tissues of F. grandis. The technique of one dimensional gel electrophoresis (1D-SDS-PAGE) was used to assess the effects of tissue sampling, flash frozen in liquid nitrogen versus immersion of fresh tissue in RNA later, for five tissues, liver, skeletal muscle, brain, gill, and heart, followed by LC-MS/MS to identify protein bands that were differentially stabilized in gill and liver. The study shows that, in F. grandis, the preferred method of preservation was tissue specific. xi Chapter 4 focuses on the use of advanced 2DE-MS/MS to characterize the proteome of multiple tissues in F. grandis. Database searching resulted in the identification of 253 non-redundant proteins in five tissues: liver, muscle, brain, gill, and heart. Identifications include enzymes of energy metabolism, heat shock proteins, and structural proteins. The protein identification rate was approximately 50 % of the protein spots analyzed. This identification rate for a species without a sequenced genome demonstrates the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.

Reported in this dissertation are the results of investigations performed at the New Orleans Center for Mass Spectrometry at the University of New Orleans. The projects that are detailed in the coming pages take on a variety of subjects, but a common thread is that each employs matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to solve a problem. Fundamental aspects of MALDI in-plume ionization are implicated in the introduction of a newly developed electron-transfer secondary ionization matrix. The remaining projects are related to the ever expanding field of proteomics. Mass spectrometry was used to investigate the site specificity of a newly developed bilin-lyase enzyme, a new approach was developed to distinguish between A-ring and D-ring attachment of bilins, and F. grandis protein expression pattern was investigated in several tissues. All obtained results were acquired using a MALDI TOF/TOF mass spectrometer. The sensitivity, mass accuracy, mass resolution and the ability to perform collision induced decomposition (CID) experiments were all valuable features that served to raise the quality of data, and thereby improved the detail of inferences to be drawn for the different projects.

<div>Following the 2010 Deepwater Horizon oil spill, extensive research has been conducted on the toxicity of oil and polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. The location and timing of the Deepwater Horizon surface slick coincided with the spawning seasons of many important pelagic and estuarine fish species. As such, there has been particular emphasis placed on the effects of PAHs on sensitive life history stages in fish, such as the embryonic and larval periods. Additionally, the spill occurred throughout the spring and summer months which, in estuaries, are marked by regular fluctuations in abiotic environmental factors such as dissolved oxygen, salinity, and temperature. Until recently, there has been little work done to elucidate the combined effects that PAHs from oil spills and adverse environmental conditions (hypoxia, increased salinity, and elevated temperatures).</div><div>Work presented in this dissertation uses next generation sequencing technology (RNA Seq) to determine differential gene expression in larval estuarine teleosts following exposure to adverse environmental conditions and PAHs. Downstream canonical pathway and toxicological function analysis were then applied to the identified differentially expressed genes (DEGs) to predict cardiotoxic responses at the organismal level. To verify the predicted responses, a phenotypic anchoring study was conducted and identified a cardiotoxic phenotype (pericardial edema) and reduced cardiac output in embryos exposed to oil. Finally, the mechano-genetic interplay governing the morphological development of the teleost heart was investigated and correlations between developmental gene expression and blood flow forces within the cardiovascular system were identified.</div>