Doctoral Dissertations

Date of Award

12-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

T. Wayne Schultz

Abstract

Physiological, developmental, and metabolic adaptations have been documented in a number of different trophic levels. It is often unclear whether a population has "acclimated" due phenotypic change or "adapted" due to actual genotypic change. While adaptation is a genetic change resulting from natural selection pressures, acclimation is a phenotypic change that results from an outside stressor. The experiments described here have expanded previous work which showed that responses of Tetrahymena pyriformis populations exposed to chemicals acting by the nonpolar narcosis mechanism of action differ based on the hydrophobicity of the chemicals. This response varied from concentration dependent growth rate for hydrophilic chemicals to a concentration dependent lag phase for hydrophobic chemicals. This previous work also suggested that T. pyriformis acclimates to sublethal levels of hydrophobic chemicals acting by the nonpolar narcosis mechanism of action. Experiments comparing the response of naive and previously exposed populations were conducted using a hydrophilic (acetone) and a hydrophobic (2-decanone) representative. Results demonstrated that T. pyriformis acclimated to 2-decanone but not acetone. As both a comparison and a validation of the growth kinetics assay, experiments were conducted using two electrophilic chemicals that have been shown to exhibit Michael-type acceptance. These two soft electrophiles, 3-butyn-2-one and trans-2-nonenal, have 1- octanol water partition coefficients (low Kow) similar to acetone and 2-decanone. Concentration dependant death was seen in the initial inoculum of T. pyriformis at threshold concentrations. Additionally, the low log Kow chemical was shown to be more potent than the trans-2-nonenal. The possibility for mixed mechanism of narcosis and electrophilicity was shown for the trans-2-nonenal. The model chemical used in the remainder of experiments was the classic nonpolar narcotic, 1-octanol. Acclimation was shown to occur in the presence of 1-octanol. Four possibilities were identified to explain the observed acclimation response 1) genetic adaptation results in a resistant population; 2) T. pyriformis quickly biodegrades the chemical; 3) lack of bioavailablity; 4) T. pyriformis contain an endogenous biochemical adaptation system which can quickly cause cellular changes resulting in acclimation. Biodegradation has been measured as total extractable 1-octanol, which did not change during the duration of the experiment. Bioavailability was measured using the solid phase microextraction techniques. There was a 25-30% decrease in freely available 1-octanol due to presence of protein components in the proteose peptone based growth medium. This decrease was constant across concentrations of 1-octanol tested. Using T. pyriformis densities that varied over 2.5 log unit, there was a decrease in bioavailable 1-octanol with increasing population densities. Although this decrease was observed, for the densities of T. pyriformis used in the growth assays, freely available 1 -octanol was constant. It is hypothesized that acclimation to toxic stress by T. pyriformis is a result of alterations in the microenvironment of the membrane, the site of action for chemical acting by nonpolar narcosis. Population growth experiments were performed in tandem with quantitative molecular analysis of fatty acid content of the pellicle layer of T. pyriformis. A control, solvent control, and three sublethal concentrations of 1-octanol were tested. The relative % of the fatty acid methyl esters, FAMEs, 16:0 and 18:0, increased with exposure to 1-octanol (fatty acid notation is carbon number of acyl- chain: number of π-bonds). Conversely, with exposure there was a decrease for the following FAMEs: 16:1, 18:1, and 18:2Δ6,11. The overall decrease in the number of π- bonds is thought to be related to a net decrease in overall fluidity. Additionally, there was an increase in the ratio of 16/18 carbon FAMEs implicating physical accommodation of the compound within the membrane.

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