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.
We studied a range of development stages in the American alligator, from eggs, to just hatched, to several months post-hatching. Our primary experimental manipulation was to alter the content of oxygen gas in the atmosphere that the eggs and alligator hatchlings experienced. In earlier work by our collaborators and coinvestigators, we have found that the size and development of the alligators is sensitive to such a variation in oxygen gas percentage. Low oxygen during development leads to much smaller hatchlings. This has some physiological relevance to the work that the lung and heart have to do to supply tissues with oxygen when it is scarcer. Historically, this is of additional interest because the atmosphere on Earth has fluctuated from oxygen levels below 16% to nearly 35%, with 20.95% being the current level. The success of alligators as an extant species dating back over millions of years may be related to their somewhat unusual four-chambered heart and sac-like lung in their vertebrate lineage. For our role as one third of a research group, we involved undergraduate students interested in physiology to analyze heart samples from these alligators, although we have several organs we can use in the future, such as liver and skeletal muscle. The students studied the contractile protein content of the heart, the "mini-motors" of muscle which determine the speed of the muscle movement, and to some degree, the efficiency of energy consumption during muscle contraction. We found changes in these proteins, called myosin heavy-chain isoforms, which are related to the growth of the alligators, but apparently not to the oxygen content of the atmosphere. This is an indication that the alligators have hearts which are sufficiently resilient to handle low and high oxygen contents, which again speaks to the success of this species in the fossil record. We also found that an oxygen storage protein, myoglobin, may be increased in the groups of alligators raised in lower oxygen – this may be a physiological compensation mechanism for these alligators, enabling them to survive even lower oxygen atmospheres.
