Publication Date

2009-08-06

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Marine Biology and Fisheries (Marine)

Date of Defense

May 2009

First Committee Member

Patrick J. Walsh - Committee Chair

Second Committee Member

Jack W. Fell - Committee Member

Third Committee Member

Robert K. Cowen - Committee Member

Fourth Committee Member

Douglas L. Crawford - Committee Member

Fifth Committee Member

Paul M. Anderson - Outside Committee Member

Abstract

Molecular aspects of nitrogen metabolism in vertebrates is an interesting area of physiology and evolution to explore due to the different ways in which animals excrete nitrogenous waste as they transition from an aquatic to a terrestrial lifestyle. Two main products of nitrogen metabolism in fishes are ammonia and urea. Ammonia is produced during protein catabolism and build up of ammonia is toxic. Some aquatic vertebrates convert ammonia into a less toxic compound urea via de novo synthesis through the ornithine-urea cycle (O-UC). Five enzymes are involved in the O-UC: carbamoyl phosphate synthetase (CPS), ornithine carbamoyl transferase (OCT), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (ARG). An accessory enzyme, glutamine synthetase (GS) also participates in the "fish-type" O-UC. Teleosts excrete ammonia passively over their gills into the aquatic environment. The teleost, Opsanus beta, has been shown to increase urea production after 48 hours of crowding. This thesis explored how crowding stress affected nitrogen metabolite levels of ammonia and urea and O-UC gene expression and enzyme activity in O. beta. Lungfishes while in an aquatic environment avoid ammonia toxicity by releasing excess ammonia across their gills, but when stranded on land they produce urea through the O-UC. Urea production via the O-UC has a metabolic cost of at least four ATP molecules. This thesis explored the response of a lungfish, Protopterus annectens, to six days of aerial exposure and re-immersion conditions by measuring concentrations of O-UC mRNA expression and enzyme activity and nitrogen metabolites ammonia and urea. CPS acts as the entry point to the O-UC and based on enzymatic studies, most aquatic vertebrates utilize one isoform of this enzyme (CPSIII) while terrestrial vertebrates utilize a different isoform of this enzyme (CPSI). Lungfishes are a particularly interesting group of air-breathing fishes, not only because of their link to the origins of tetrapods, but also because CPS I may have originated within this group. Both CPS III and CPS I have been enzymatically described within this group. This thesis uses phylogenetics to investigate how CPS nucleotide sequences in lungfishes evolved compared to other vertebrates.

Keywords

Coordinated Induction Of Ornithine-urea Enzymes; Liver; Lungfish Phylogenetics; Metabolic Pathway; Vertebrate Water-land Transition; Messenger RNA Production; Protein Production; Carbamoyl Phosphate Synthetase Evolution

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