Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Department

Biological Sciences

First Advisor

Brad S. Seibel

Abstract

Trimethylamine oxide (TMAO) was first described in marine organisms as an osmolyte, involved in the balance of water and solutes. After its discovery, it was found to be part of a subset of osmotic constituents termed counteracting solutes. These solutes exhibit stabilizing properties and can preserve protein functionality against biological and environmental perturbations. TMAO acts as a universal stabilizer, protecting macromolecular structure and function in response to numerous stressors, including urea destabilization, hydrostatic pressure, temperature and salinity. The studies presented in this dissertation address the regulatory and environmental factors affecting TMAO accumulation.

Both exogenous and endogenous sources are involved in the maintenance of TMAO. Exogenous TMAO accumulates through absorption from the diet while endogenous TMAO is synthesized from dietary or cellular precursors with the flavincontaining monooxygenase trimethylamine oxidase (TMAoxi). Species without a physiologically relevant synthetic capacity are hypothesized to rely entirely on dietary contributions for accumulation. Chapter 1 examines the necessity of an exogenous TMAO source on long-term maintenance in elasmobranch species with and without the ability for endogenous synthesis. These data show that presence or absence of TMAoxi cannot be used as a proxy to determine the importance of dietary TMAO on prolonged conservation. It seems that all species, regardless of synthesizing potential, rely to an extent on contributions from the diet.

Chapter 2 further examines the regulatory factors affecting TMAO. This study provides evidence for endogenous production via an understudied synthetic pathway whereby TMAO is accumulated as a byproduct during lipid storage. The existence of this pathway is supported by a correlation between TMAO content and total lipid in a variety of Hawaiian mid-water fishes. The regulatory role of evolutionary relatedness on accumulation potential is also addressed in this chapter. Phylogenetic independent contrasts (PIC) showed no relationship between phylogeny and TMAO content across 27 species spanning nine orders. This suggests that environmental factors impart a larger influence on TMAO retention than evolutionary history.

Chapter 2 goes on to examine TMAO’s role in combatting the environmental stress associated with increasing hydrostatic pressure. TMAO was shown to increase with increasing depth of occurrence across all species of Hawaiian mid-water fishes studied. These data support previous reports of TMAO accumulation as an environmental adaptation to combat the destabilizing effects of elevated hydrostatic pressure.

Chapter 3 explores TMAO’s ability to counteract environmental fluctuations in temperature. Previous in vitro studies showed intracellular transport and accumulation of TMAO with increasing temperature in elasmobranch red blood cells. Further, this was shown to suppress the traditional heat shock response of heat shock protein 70 (HSP70) upregulation. However, we saw no increase in plasma or tissue TMAO in response to elevated temperature for two shark species in vivo. Either mechanisms established in vitro are not applicable at the organismal level or additional regulatory factors are limiting TMAO accumulation.

Lastly, a brief study examining regulation of TMAO through ontogeny in an elasmobranch species, Squalus acanthias, is presented in the Appendix. Pups of this species exhibit low levels of urea and TMAO, their two primary osmolytes. However, total osmotic pressure is maintained at adult levels. Therefore, a shift in the osmotic milieu occurs sometime between birth and adulthood. These findings are in contrast to those reported for the little skate, Leucoraja erinacea, which expresses adult levels of these osmotic constituents early in development. These data point to divergence in the early osmoregulatory strategies of differing elasmobranch groups.

In the enclosed chapters, key objectives regarding the regulatory and environmental factors influencing TMAO are addressed. Specifically, this research examines how contributing sources, evolutionary restrictions and environmental stress affect TMAO accumulation. These studies elucidate TMAO’s multifaceted role in marine organisms and provide insight into the factors regulating its adaptive potential.

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