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.
This project was designed to study the role of atmospheric oxygen on the growth and function of the skeleton in the American alligator. Alligators are an important species to study because they are thought to be biologically similar to the ancestors of dinosaurs. One of the major unanswered questions about alligator biology is how long-term changes in oxygen availability may alter growth and function of the skeleton. Across geological time, beginning about 350 million-years-ago, atmospheric oxygen has varied considerably (Figure 1). The minimum oxygen level may have been only about 75% of present level, and the maximum is thought to be 150% of the present level. Throughout this time, a wide diversity of animals, including crocodiles, dinosaurs, turtles, and mammal ancestors, originated and diversified. Much research in recent years has focused on trying to understand the physiology of those early representatives of modern groups. Because only skeletons remain, the micro-scale structure of bone of these animals is studied to estimate these extinct animals' physiology. An important missing piece of this puzzle is how available oxygen in the atmosphere over several years might alter the signal of physiology in bones. To address this gap in our knowledge, we studied skeletons of alligators that were raised from eggs to up to 2 years in one of five treatment groups, one with less oxygen than present, three with more oxygen than present, and one control group with the same oxygen level as present. At six time points during the study, skeletons were collected for further study. We dissected out the thigh bone from each animal and studied its size and shape, each of which are informative about how the skeleton grows and responds to loading as might be encountered during locomotion (Figures 2 and 3. In contrast to other studies on growth in altered oxygen levels, we found no differences in growth between treatment groups across 2 years of growth (Figure 4). A key difference between this study and previous ones is the length of the study (days or weeks vs. two years). Two explanations for our results are possible. First, alligators might be insensitive to altered oxygen over the long term, even though they might be susceptible over the short time. Second, alligators might be able to alter their physiology so that they are not sensitive to altered atmospheric oxygen. We then tested the mechanical abilities of the bones. Here we also found no effect of oxygen treatment (Figure 5). However, we did discover that alligators exhibit a very unusual pattern of bone growth (Figure 2), which results in very thick bones, similar to what is seen in other aquatic vertebrates like whales, manatees, and penguins. We hypothesize that this unusual growth pattern is associated with their aquatic lifestyle. The impacts of the project extend beyond the science. This project has supported two young scientists, one a Master of Science in Biology student, and one a technician who is preparing to pusue a doctorate in Biology. Both students are pursuing careers in which they will train and inspire the next generation of scientists through research, teaching, training, and outreach. The results of this project have been broadly disseminated, both to the scientific community through conference presentations and to general audiences. Project personnel have spoken at regional, national, and international conferences as well as to interested groups of undergraduates at numerous universities. This project has implications not only for directly interested groups of scientists but more widely to those interested in understanding the role of the natural environment in the process of evolution and in understanding how alligators grow. Results of this project have been incorporated into teaching materials to help educate developing scientists in biology.
