Thesis (M.A.)--University of California, 1928. / Includes bibliographical references (p. 42-50).

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 87-92.

The Gulf killifish Fundulus grandis is a euryhaline baitfish species with commercial aquaculture potential. The low salinity and freshwater tolerance of early life-stages of F. grandis were evaluated to determine the potential for inland culture and guide production protocols. Egg fertilization and embryo survival decreased in fresh water, but freshwater incubation improved larval freshwater tolerance. In juveniles, survival and growth were reduced in fresh water from 2-6 weeks post-hatch but not from 7-11 or 12-16 weeks post-hatch. Na+/K+-ATPase activity and whole body Na+ content were elevated in low salinity treatments of the 7-11 weeks post-hatch age group, indicating substantial osmoregulatory ability at that age. RNA expression data from this age group indicated that freshwater acclimation is accompanied by an initial up-regulation of carbonic anhydrase followed by prolonged up-regulation of Na+/K+-ATPase. By 12-16 weeks post-hatch, F. grandis osmoregulatory ability is well developed with minimal differences among osmoregulatory data.

This thesis examined the ionoregulatory physiology of freshwater-adapted mummichog (𝘍𝘶𝘯𝘥𝘶𝘭𝘶𝘴 𝘩𝘦𝘵𝘦𝘳𝘰𝘤𝘭𝘪𝘵𝘶𝘴) 𝘪𝘯 𝘷𝘪𝘷𝘰 and its interaction with acid-base regulation. Under control conditions (water [NaCl] ≈ 0.7-1 .0 mmol· L⁻¹, [Ca²⁺] ≈ 0.1 or 1.0 mmol· L⁻¹), Na⁺ turnover was vigorous with a positive Na⁺ balance maintained, whereas unidirectional Cl⁻ influx was virtually zero resulting in a slight negative Cl⁻ balance. Michaelis-Menten analysis revealed a low affinity, high capacity Na⁺ uptake mechanism that was independent of both Na⁺ efflux and ammonia excretion. Cl⁻ uptake started at higher water [NaCl] levels ( > 2 mmol • L⁻¹) but did not saturate within the freshwater [NaCl] range, indicating a completely different uptake mechanism, independent from Na⁺ influx. Using both internal and environmental manipulation, Na⁺ uptake was found not to be coupled to ammonia excretion. Instead, a link with acid excretion (Na⁺ /H⁺ exchange or Na⁺ channel/H⁺ -ATPase coupling) remains possible but could not be confirmed. There was no evidence for the presence of a Cl⁻/HC0₃⁻exchange mechanism in the gills. However, mummichog were capable of differentially manipulating Na⁺ and Cl⁻ efflux components as an additional response to an internal acid-base disturbance. This ability and the suggested Na⁺ uptake/acid excretion coupling indicate that mummichog resemble other freshwater fish in that an iono/acid-base relationship exists. In these studies, the use of the Strong lon Difference Theory as a means of assessing acid-base balance through the measurement of differential Na⁺ and Cl⁻ fluxes proved to be acceptable and practical alternative to the measurement of acid-base fluxes by traditional titration methodology. Finally, whole-body Ca²⁺ uptake was investigated using a recently developed technique for small fish. Ca²⁺ uptake by the mummichog involves a carrier-mediated step as revealed by saturation of uptake as external [Ca²⁺] increased. Inhibition of uptake by external La³⁺ but not Mg²⁺ suggested that apical Ca²⁺ channels are involved in the uptake process but are not voltage-gated. Chronic exposure to low Ca²⁺ water resulted in a stimulated Ca²⁺ uptake, most likely in response to depletion of internal Ca²⁺ levels whereas chronic exposure to high Ca²⁺ did not elicit any changes in uptake. This thesis revealed that the freshwater-adapted mummichog does share certain ionoregulatory qualities with other freshwater fish but at the same time possesses unique characteristics which may reflect its euryhaline nature / Thesis / Master of Science (MS)

This study was made to further define the trophic dynamics of Fundulus notatus by determining its ration composition under natural conditions, measuring feeding selectivity under various laboratory conditions of prey-species composition and availability, and determining the efficiencies with which F. notatus utilizes ingested chironomid larvae.

Hypoxia in the marine environment is a growing environmental concern, and can have profound impacts on organisms. This dissertation seeks to understand the physiologically induced changes in gene expression, the relationship between gene expression and metabolism, and how these parameters vary among populations, in response to hypoxic stress. By comparing evolved intraspecific variation in gene expression and physiological parameters among populations from multiple regions in the Gulf of Mexico we seek to determine the physiologically induced changes that are essential to hypoxic survival. First, whole body metabolism, measured as oxygen uptake, was profiled across seven decreasing oxygen concentrations. Metabolism and the critical oxygen tension (PO2crit) were compared between populations from across the Gulf of Mexico. This study demonstrated a significant interaction of body mass with the hypoxic response. Additionally, populations only differed in their metabolism at the lowest oxygen concentration, 1.8 kPa. PO2crit did not differ between populations, but was body mass dependant. Next, the effects of hypoxia on gene expression were examined. These studies examined the effects of hypoxia on gene expression over time and at different hypoxic doses, utilizing a 384 gene microarray. In the first studies individuals were subjected to 0, 4, 8, 12, 24, 48, or 96 hours of hypoxia. Different genes had different times for peak gene expression, with most changes occurring after 96 hours of exposure. However, only 14 genes had significant changes in gene expression. To determine the effect of differing hypoxic dose, individuals were exposed to normoxia, 7.8 kPa O2 (moderate hypoxia), or 1.8 kPa (severe hypoxia) for 4 or 48 hours. Sixty-nine genes had significant changes in gene expression for either dose or time. To elucidate the relationship between effect of time and dose, genes were examined for dose response within each time. The maximum number of changes occurred at 1.8 kPa after 48 hours of exposure. Interestingly different sets of genes had changes in gene expression at either 7.8 or 1.8 kPa. Finally, to ascertain the difference among populations, for thousands of genes, individuals from six populations of Fundulus grandis were exposed to hypoxia (1.8 kPa) for 4 or 96 hours. Hypoxia had a significant effect on the expression of 609 genes, while population affected the expression of 355 genes. Genes with significant differences in expression among populations reflect geographic separation. For the 59 genes with significant differences in expression for both hypoxia response and population, shared hypoxic histories appears to be more important than simply the neutral patterns expected with geographic distance. The majority of significant changes for the 609 hypoxia responsive genes take place after 96 hours of hypoxia exposure. This research demonstrates that F. grandis cope with hypoxia through changes in metabolism and gene expression. Overall, the response to hypoxia is dependent on an individual's size (body mass), the ambient oxygen concentration, and the duration of hypoxia exposure. Additionally, there appear to be some differences between populations with differing exposure history to hypoxia in the Gulf of Mexico.

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.

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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.

La présence d’hybrides asexués gynogénétiques entre Fundulus diaphanus et F. heteroclitus à deux sites en Nouvelle-Écosse (Porter’s Lake et Saint-Mary’s River) est documentée dans la littérature. Un échantillonnage aléatoire de Fundulus à douze stations le long du gradient de salinité de Porter’s Lake et le génotypage à l’aide de huit marqueurs microsatellites a permis de découvrir la présence d’un clone principal et de ses dérivés mutationnels, constituant ensemble 97% des hybrides. D’autre part, une méthode d’identification morphologique des F. diaphanus, des F. heteroclitus et des hybrides basée sur un algorithme de vraisemblance maximale a été élaborée pour utilisation sur le terrain, permettant une diagnose rapide et efficace à plus de 90%. Une grande variabilité morphologique des hybrides a été observée à l’aide de la morphométrie géométrique. Finalement, la vaste répartition d’un clone principal et de ses dérivés mutationnels, morphologiquement variables, laisse supposer la présence un génotype généraliste à Porter’s Lake. / Presence of asexual hybrids between Fundulus diaphanus and F. heteroclitus at two sites in Nova Scotia (Porter’s Lake and Saint-Mary’s River) has been documented in the literature. Random sampling of Fundulus along the salinity gradient of Porter’s Lake and genotyping using eight microsatellite markers revealed one main clone and its mutational derivatives in the lake, representing 97% of hybrids encountered. Also, a novel method of morphological identification of F. diaphanus, F. heteroclitus and hybrids based on a maximum likelihood algorithm has been elaborated for fieldwork purposes, allowing a quick, over 90% efficient, way to diagnose the three Fundulus types. Morphological analyses performed using geometric morphometrics also revealed that hybrids were no less variable morphologically than parental species. Finally, the presence of one main clone in different salinities and an important morphological variability suggest the presence of a General-Purpose Genotype in Porter’s Lake.