The atmosphere serves as a source of oxygen (O2) for all organisms that rely on O2 to power their metabolism. Today, the level of atmospheric O2 is approximately 21%, however over the past 550 million years O2 levels may have risen as high as 30-35% and dropped as low as 12%. Geologists have long recognized that large-scale fluctuations in atmospheric O2 would have had significant effects on the physiology of contemporary organisms and have hypothesized that changes in the ancient atmosphere resulted in significant behavioral, physiological and ecological adaptations. This collaborative research will recreate ancient atmospheric O2 conditions in order to investigate the effects of chronic hypoxia (low oxygen) and hyperoxia (high oxygen) on embryonic development, physiological function and the skeletal system of the American alligator. Alligators are a "cold blooded" representative of a large vertebrate group, Archosauria, which also includes dinosaurs and bird. Archosaurs originated in the Late Permian (ca. 280 million years ago) when the atmosphere was O2 rich and experienced the Late Triassic (circa 220 mya) when the atmosphere was O2 poor. Alligators obviously survived (and thrived) despite large-scale fluctuations in atmospheric O2. This will be the first such study in a post-embryonic vertebrate animal engaging in a wide variety of natural behaviors (rest, voluntary and forced activity, recovery from exercise, digestion, fasting) chronically exposed to different O2 levels. Alligator eggs will be incubated under six treatments: hypoxia (12 and 16%), normoxia (21%) and hyperoxia (25, 30 and 35%). Developmental progress, incubation time, hatching success and whole- embryo metabolic rate will be recorded. After hatching, some alligators will continue growing under the same conditions as during incubation. Others will be transferred between treatments, in order to determine whether (and how) the O2 environment of the embryo constrains the anatomy and physiology of the hatchling. This collaborative project will bring together three laboratories with different scientific emphases - whole-animal physiology, bone histology and molecular biology - and will broaden our understanding of how interactions of physiological, anatomical and biochemical processes are integrated to determine overall organismal performance. This project will train undergraduates, graduates and postdoctoral fellows, and provide a framework within which to analyze systems in terms of environmental influences on organismal form and function, and to place research results within developmental and/or evolutionary trajectories. Results from this project will find direct industry application by improving alligator farming methods, and the wealth of data and tissue samples generated by our experiments will be shared with other researchers via the Alligator Tissue Bank at UC Irvine. Finally, this project will include a strong outreach component to both K-12 students and their teachers, primarily from schools representing high-minority and low socioeconomic areas in Southern California. The integrative nature of the project will allow us to design inquiry-based demonstrations and educational modules, using raw data generated from experiments, which address several life and earth science educational content standards.
The earth’s atmosphere serves as a source of oxygen (O2) for all organisms, which rely on aerobiosis (biochemical pathways using oxygen) to power their metabolism. At present, the level of atmospheric O2 is approximately 21%, however over the past 550 million years O2 levels may have risen as high as 30-35% and dropped as low as 12% (Berner, 2006). Geologists have long recognized that large-scale fluctuations in atmospheric O2 could have had significant effects on the physiology of contemporary organisms (McAlester, 1970; Erwin, 1993). Graham et al. (1995) hypothesized that a steady rise in atmospheric O2 spanning the mid-Devonian to mid-Permian periods (approximately 150 million year period) allowed significant diversification of behavioral, physiological and ecological adaptations in animals. Huey and Ward (2005) suggested that the decline in atmospheric O2 at the end of the Permian, concurrent with global warming, might have caused mass-extinctions through habitat reduction and fragmentation of populations. However, despite repeated calls for more "paleophysiological studies, from both a fossil interpretation standpoint and a modern experimental standpoint" (Berner 1999; Berner et al., 2003), few experimental studies on chronic exposure conditions mimicking the hypothesized paleoatmosphere have been conducted (Berner et al., 2007). Only recently, projects have recreated the paleoatmospheres in the laboratory; a sort of atmospheric time machine (ATM). These studies are indicating that exposure to chronic hypoxia and hyperoxia affect growth and metabolism of insects (Harrison, et al., 2006; Kaiser et al., 2007) and vertebrate animals (Vanden Brooks, 2004; Chan and Burggren, 2005), supporting the notion that fluctuations in atmospheric oxygen may have been an important stimulus driving the evolution of animals. Based on this background, although we cannot directly measure physiologic function of extinct species, we can learn how chronic changes in environmental O2 affect the developmental trajectories and physiological function of living vertebrate animals and use these findings to provide inferences about ancestral species. Consequently, the goals of this research project were to investigate development, growth, skeletal architecture, metabolism and cardiopulmonary physiology of the American alligator living under chronic environmental hypoxia (low O2) and hyperoxia (high O2). In addition the project was used to train undergraduates, graduate students and postdoctoral fellows in experimental hypothesis testing research and to use comprehensive and integrative physiological techniques in order to understand organismic-, tissue-, cell- and molecular-level responses to environmental change. The alligator was chosen as a model species because it is an ectothermic representative of the large clade Archosauria, which encompasses all crocodilians, phytosaurs (ancient crocodilian-like reptiles), pterosaurs (flying reptiles) and dinosaurs (including birds). Based on the completed experiments, neither metabolic rates nor growth rates of alligators (embryos, hatchlings and juveniles up to two year old), were significantly altered by chronic hypoxia (16% O2) or hyperoxia (26, 31 and 36% O2). Only severe hypoxia (12% O2) has a significant effect on metabolic rate (significantly elevated) and on growth rate (significantly depressed). Neither resting nor maximal (elicited by treadmill exercise) rates of oxygen consumption were significantly different between treatment groups. Growth of individual organs (liver, lungs, kidneys, select skeletal muscles) follows a similar pattern (hypoxia and hyperoxia had little effect). The heart, however, appears to be affected by atmospheric oxygen level, with the right ventricle increasing in mass with decreasing %O2. Hematocrit (number of red blood cells) shows an inverse pattern, with a significant decrease with increasing %O2. The study indicates that the heart is the most sensitive organ in response to changes in oxygen levels. Interestingly, in the alligator, the cardiac outflow tracts are remodeled in response to chronic exposure to %O2, becoming less developed in animals reared under chronic low oxygen levels. These findings led us to speculate as to the role of atmospheric hypoxia in driving or contributing to the evolution of the mammalian and avian heart. These experimental findings directly contribute a deeper understanding of how living vertebrates respond to low oxygen levels and also contribute to the field of paleobiology. Overall, the experimental results of this project can be used to make inferences about the role of atmospheric oxygen levels on the evolutionary trajectories of vertebrate animals.
