A COMPARISON OF CARBONIC ANHYDRASE AMONG INVERTEBRATES, WITH A FOCUS ON CEPHALOPODS

Carbon dioxide (CO2) is both an unavoidable waste product of aerobic carbohydrate metabolism and a fuel source for autotrophic and chemoautotrophic organisms. At biological pH, CO2 rapidly reacts with interstitial and intracellular water to form carbonic acid (H2CO3), that then dissociates into protons (H) and bicarbonate (HCO3). While CO2 and H readily diffuse across biological membranes, the majority of the CO2 in living tissues is in the membrane-impermeable form HCO3. The enzyme carbonic anhydrase (CA) catalyzes the reversible hydration of CO2 with water to maintain an instantaneous equilibrium between these chemical species. CA is not only central to the transport and excretion of CO2 in animals (or uptake in autotrophic organisms), but is also indirectly involved in important physiological processes, such as osmoregulation and acid-base balance. The multiple functions of CA are a result of multiple isoforms that are localized to specific subcellular compartments/fractions. Furthermore, the level of CA activity in an organism can be induced to change in response to conditions in the ambient environment, and may also reflect the metabolic rate of the organism. This enzyme has been studied in cell and tissue types from numerous organisms, but has never been systematically characterized in squids. This dissertation examined CA activity in gill and mantle muscle among several cephalopod species in terms of aerobic mass-specific metabolic rates (MR), evolutionary relationships, and environmental conditions. It also compared the protein-specific activity of CA in the respiratory tissue reported in the literature for a broad array of invertebrates. The CA activity in gill and mantle muscle tissue from three squid species was measured to examine whether differences in activity may be related to phylogenetic relationships or environmental adaptations. The three squid species, Dosidicus gigas, Lolliguncula brevis, and Doryteuthis pealeii have similar MR but endure different physiological demands due to their respective environmental conditions. The largest member of family Ommastrephidae, Dosidicus gigas, undergoes diel vertical migrations into a well-defined oxygen minimum zone in the eastern Pacific. The brief squid, Lolliguncula brevis, is the only squid species that inhabits the wide-ranging abiotic conditions of estuarine waters. This species is in the same family as Doryteuthis pealeii, yet the latter requires narrower environmental parameters. For all three species the total CA activity was greater in gill tissue than in mantle muscle, but the activity in each tissue was statistically the same between these species. The distribution of CA isozymes within the subcellular compartments, however, was more similar between D. gigas and L. brevis, species that chronically endure variable conditions, than between more closely related L. brevis and D. pealeii. Reflecting the similar MR of these three species, total CA activity in gill scaled independent of both mass and MR, however there was a significant relationship in mantle muscle. The findings suggest that while total CA activity is reflective of whole organism, environmental conditions seem to affect the subcellular distribution of this enzyme. Additionally, it appears that the primary function of CA in cephalopod gills is towards the removal of the large amounts of CO2 produced in these muscular, highly active animals. Broadening the comparison of CA activity among cephalopods, I examined the CA activity in gill and muscle tissue from cephalopod species at different depths to 1) further test the visual interaction hypothesis and 2) examine the relationship between CA activity and metabolic rate. The visual interactions hypothesis states that the decreasing metabolic rates with increasing depth distribution among visually-orienting oceanic species is due to relaxed selection for strong locomotory capacity because the diminished light levels result in less frequent predator-prey interactions. The CA activity in gills and gill mass each declined significantly with increasing depth. The protein concentration in both gill and mantle muscle also showed a significant decline with depth distribution. The CA activity in both gill and mantle muscle exhibited a strong, direct correlation with metabolic rates among cephalopods. These findings are in agreement with other studies that have tested the visual interactions hypothesis. Additionally, the activity of CA is clearly correlated with aerobic mass-specific metabolic rate among cephalopods. To examine factors that affect CA activity once the influences of animal mass and metabolic rates are removed, I compared the protein-specific activity of CA in the respiratory tissues of 67 invertebrate species across four phyla. The data was obtained from published values of specific CA activity that met specific inclusion criteria. The specific CA activity values for all species sampled was analyzed in terms of taxonomic group, type of respiratory tissue, habitat, symbiotic relationships, and type of homogenate used in the assay. A subset of the data was utilized to examine residual correlations between specific CA activity and estimates of animal mass and metabolic rates. The weighted mean masses and metabolic rates within each family were calculated from values obtained through a second literature search. Using this truncated dataset only bivalves exhibited a correlation between specific CA activity and metabolic rate. The results suggest that, once difference in mass and MR are accounted for, the physiological demands placed on an organism to survive in a given habitat seem to be the driving factor underlying CA activity. This work is the first to systematically analyze CA activity in cephalopods, and contributes to the growing body of knowledge about CA activity in invertebrate respiratory and muscle tissues. This research also broadens scientific understanding of the relationships between CA activity and whole animal metabolic rates, the impacts of an organism's environment, and differences among various invertebrate taxa. ACKNOWLEDGEMENTS I would like to take this opportunity to dedicate this work to some important people in my life. To my daughter Caly, I never would have achieved this without your love. You inspired me to persevere through some really tough times more than you will ever know. A large part of my drive to complete this degree was to show you that you can accomplish anything you set your mind to. My grandfather, Ed Nyack, showed me that more can be conveyed in a single glance than in one thousand words. I miss you every day grandpa. To my fiancee Brooke Somers, I am ever so thankful that you found me, for always looking on the bright side, and being the repetitive 'you can do it' voice in my ear. Finally, Dorothy A. Cashdollar RN, my mother, is the strongest person I have ever known. I have no doubt that I never would have accomplished this milestone without her steadfast encouragement and tireless ear. This research would not have been possible without the knowledge, guidance, and TREMENDOUS patience of my major advisor, Brad A. Seibel. He taught me how to be a better writer and critical thinker, and he honed my skills as a scientist. Thank you, Brad, for standing by me though some rough times, and for both pushing me and giving me latitude as the occasion required. I would like to thank Ray Henry for offering to be on my committee, for his mentorship, and for lending his expertise throughout the course of this research. I am incredibly thankful for all of Marta Gomez-Chiarri's helpful advice, insight, and support. I am also grateful to Jackie Webb for her caring, straight-forward approach, constructive feedback, and keen attention to detail. Finally, my profound thanks to