Abstract
Marine fish must constantly work to maintain proper salt and water balance, as they live in an environment with a salt concentration approximately three-fold higher than their body fluids. Living in such a hyperosmotic environment results in the continual passive loss of water and gain of salts, which must be compensated for to maintain osmotic balance. Replacing lost water is particularly challenging, as the only source of water available is seawater. Marine teleosts possess a suite of adaptations in order to extract water from imbibed seawater, one of which is the precipitation of inorganic CaCO3 in the intestinal lumen, which lowers luminal osmotic pressure by as much as 100 mOsm /kg, allowing for increased water absorption. This precipitation reaction is crucial for the survival of marine fish, as they would be unable to extract sufficient water from the seawater to counteract passive water loss without the reduction of luminal osmotic pressure arising from production of carbonate mineral. Further, this precipitation plays a substantial role in oceanic inorganic carbon cycling, as mineral produced in the teleost intestine is excreted into the environment, which accounts for a conservatively estimated 3 to 15% of annual global oceanic CaCO3 production. Although there have been numerous studies investigating how the intestinal fluid chemistry is regulated, and the impacts this may have on CaCO3 precipitation, no work has aimed to determine if the precipitation reaction itself may be directly regulated in vivo. The work in this dissertation reveals that teleost intestinal precipitates form in conjunction with an organic matrix, which contains proteins that are capable of regulating CaCO3 production in vitro. The effects of these proteins on calcification are highly dose-dependent, with low concentrations leading to increased CaCO3 production, where high concentrations completely inhibit precipitation. Mass spectrometry analysis completed on isolated precipitate matrix derived from both Gulf toadfish (Opsanus beta) and European flounder (Platichthys flesus) led to the identification of over 200 proteins found in association with the forming mineral. To determine the potential functions of individual proteins in the matrix, organic matrix extracted from Gulf toadfish precipitates was fractionated in order to separate the proteins by charge. The ability of the fractions to regulate precipitation was then assessed, and the proteins in each fraction were identified, allowing for the prediction of individual protein function. To gain further insight into the functions of individual matrix proteins, a large-scale, shotgun proteomic investigation of the toadfish intestinal response to hypersalinity exposure was completed. As intestinal precipitation is known to increase during hypersalinity exposure, this experiment gave insight as to what functions the matrix proteins may play in vivo, and also allowed for numerous other questions related to intestinal physiology to be addressed. Overall, the work presented in this dissertation clearly demonstrates that carbonate mineral precipitation in the marine teleost intestine occurs in association with a partially proteinaceous organic matrix, which allows for the precise control of this process crucial to the survival of the largest group of vertebrates on earth.