L. Michael Romero - US grants

L. Michael Romero
Physiology of Stress in Wild Animals
Proposal Number 0235044

All organisms are faced with noxious stimuli from the environment (such as predators or storms). Vertebrates activate a suite of physiological mechanisms to protect themselves from these stimuli, collectively called the stress response. A vital part of the stress response is the release of the glucocorticoid steroid hormones, but even after more than 60 years of studying stress we still have only a rudimentary idea of how glucocorticoids help wild animals survive. Earlier work indicated that several free-living bird species seasonally regulate glucocorticoid release by varying concentrations of the hormone throughout the year. This proposal builds upon these results to ask the following questions: why do animals seasonally regulate glucocorticoid levels; is there a physiological consequence; and does it affect their susceptibility to chronic stress?
Answering these questions will require an integrated approach of both laboratory and field studies, such as the one proposed here. We will capture wild free-living house sparrows (Passer domesticus) and test how glucocorticoids help regulate blood glucose and triglyceride levels at different times of the year. We anticipate that glucose and triglyceride regulation will depend upon seasonal changes in glucocorticoid levels. We will also bring house sparrows into the laboratory to test whether physiological responses to chronic stress vary seasonally. We anticipate that the magnitude of the negative consequences of chronic stress will depend upon the time of year. These results will help define the physiological consequences of seasonal glucocorticoid release during stress in wild animals, thus having a far-reaching impact on our basic knowledge of the ecological validity of stress responses.
This research will also provide important training for students. Both graduate and undergraduate students will be exposed to, and learn, many important research skills in both the laboratory and field. These findings will be valuable to researchers in diverse fields including Conservation Biology, Endocrinology, and Population Biology, just to mention a few.

Many animal populations experience catastrophic population declines which are often caused by natural or human-induced changes in environmental conditions such as climate change or habitat alteration. In response to environment disasters, all vertebrate animals show a stress response that involves the secretion of the stress hormone (corticosterone/cortisol) into the blood. It is so far unclear whether and how a stress response helps individuals to survive, and whether a low or a high stress response is better. The main problem is that it is very hard to study dying animals in the wild. Here professors Wikelski and Romero make use of the fact that Galapagos marine iguanas repeatedly experience natural catastrophes (El Nino weather events) but at the same time are tame and can easily be observed as they die. The researchers will assess the stress response of individuals before a catastrophe and test what kind of a stress response helps individuals most to survive. This research will be put into the context of a 25-year data set that will be made public.

The results of this study will significantly advance the understanding of how vertebrates physiologically deal with catastrophic events in their environment. This project will also educate the public via TV shows and publications, foster scientific exchange between Latin American and US researchers and provide advanced training for undergraduate and graduate students.

Scientific Goals
All organisms are faced with challenges (such as predators or storms) from the environment. Animals mount a stress response in order to survive these challenges, but even after nearly 100 years of studying stress, there is only a basic understanding of how the stress response helps wild animals to survive. This lack of knowledge is of growing significance because man-made noxious events (such as habitat degradation or human disturbance) could cause chronic stress. Although a short-term stress response to a predator or storm is necessary for survival, chronic stress can be very harmful, and if it affects enough individuals, can cause population declines.

This project will use wild European starlings as a model to test two hypotheses: (1) That increased frequency of normal environmental events can result in chronic stress in wild animals; and (2) There will be long-term impacts on offspring whose mothers were exposed to chronic stress. Testing these hypotheses will require an integrated approach of laboratory and field studies on the physiology and behavior of both captive and free-living starlings. The ultimate goal is to create a physiological and endocrinological profile of chronically stressed wild animals. This will provide criteria for identifying chronically stressed individuals in the wild and determining the long-term impact on those individuals and their offspring.

Broader Impacts
Student participation will be vital to accomplishment of the research goals. Graduate students, undergraduate students, and a postdoctoral fellow will receive important training in how to trap animals, take samples, perform assays, write manuscripts, and other important laboratory and field skills. Conservation missions could benefit from additional information to determine whether animals are chronically stressed in the wild.

The quality of parental care influences offspring fitness. In domesticated laboratory rodents, pups that have chronically stressed mothers receive low quality care, leading pups to develop highly reactive stress responses. Affected pups will have high stress hormone levels as adults, which can have severe consequences on their future survival and reproductive output. This research examines the relationship between stressed mothers, poor maternal care, and the offspring stress response and survival in the degu (Octodon degus), a wild, free-living rodent. Degus practice a unique reproductive tactic called "plural breeding with communal care" where multiple mothers cooperate to raise each other's pups. This research also will ask whether this unique social strategy can buffer the offspring against post-natal stress. By manipulating the stress levels of mothers and measuring stress hormone levels in pups in both field and laboratory experiments, this research will provide a comprehensive description of the links between stress, development, and, ultimately, survival. This link has never been demonstrated in a wild, free-living rodent. This research will have broader impacts because it will address the link between parental care and offspring survival, which is a major unsolved problem in ecology and stress physiology. Further broader impacts of this research will include scientific outreach through a weekly blog that is incorporated into the curriculum of an undergraduate course, presentations to senior citizens and middle/high school girls, undergraduate research opportunities, and support for a young scientist completing her PhD research.

The vertebrate stress response (the physiological mechanisms involved in protecting an organism from a stressor) is a major mechanism of resilience and an important transducer of biotic interactions (between organisms), and between organisms and their non-living environment (abiotic interactions). It is astonishing, however, that even after nearly 100 years of studying stress, the overall field of stress biology lacks an integrated theory and associated models to enable quantitative and predictive research connecting the multiple mechanisms and outcomes of the stress response. This workshop will include equal numbers of researchers interested in the basic biology of stress in animals (typically funded by NSF) and researchers interested in the role of stress in healthy humans specifically (represented by researchers from NASA). NSF-funded and NASA researchers have substantial common ground. Both communities are interested in how stress biology is manifested in healthy individuals, rather than a cause of disease. However, these two communities do not typically interact owing to the conceptual and methodological constraints on the scope of their research. Both communities see an opportunity to make substantial progress in understanding this complex area of biology by sharing their perspectives and developing multidisciplinary collaborations. The cross-fertilization created by the workshop should yield novel insights about conceptual and technical challenges and opportunities, which will help guide future research in both communities. The overarching aims of the workshop will be to: 1. Identify and surmount bottlenecks that are preventing progress, and 2. Begin to develop specific research approaches to implement integrated studies. This workshop could have profound impacts on the field by facilitating the development of a new conceptual model of stress. Such a model could result in more quantitative, predictive research linking cellular and physiological stress responses with individual-, population- and species-level outcomes. The Principal Investigator anticipates publishing a peer reviewed workshop report. New avenues of research identified by the workshop participants and disseminated by the report will provide a blueprint for further study that should have a broad impact on many scientific fields.

Non-technical Abstract
The stress response is a major mechanism of resilience. However, even after nearly 100 years of studying stress we still lack a rigorous predictive model that allows us to know what the impact of stressors on individuals and populations might be. The lack of such a model is a long-standing roadblock to our understanding of the ecology and pathophysiology of stress responses. This project will create and test the first quantitative predictive model of stress pathophysiology. The foundation will be the Reactive Scope Model, a graphical model of stress. A central component of this graphical model is that prolonged activation of a stress response creates wear-and-tear. Here a mathematical model of stress based upon wear-and-tear will be constructed and integrated with laboratory studies using captive birds as models. Overall, the project will generate a new predictive quantitative model for how individuals, and by extension populations of individuals, will respond to stressors. The creation of an integrated conceptual and quantitative theory will provide the foundation to resolve long-standing questions in stress physiology and open up novel areas of research. This project will provide training of students at all levels. Specific activities will recruit women and minority students to research positions. Cross-disciplinary training will teach these students how to integrate physiological and quantitative techniques. A weekly research blog by research students will incorporate the research into an introductory Endocrinology class, thereby providing a research focus to a lecture class. The work will be disseminated through publications in the research literature and presentations to the general public.

Technical Abstract
The quantitative model will consist of a system of nonlinear partial differential equations that captures the dynamics of the Reactive Scope conceptual model. Using two established stress and stress response protocols, introduction to captivity and rotating intermittent stressors applied to captive birds, numerical solutions to deterministic approximations suggested by the Reactive Scope model will be tested against empirical experimentation. The key goal will be to understand the temporal dynamics of wear-and-tear. Predicted timing and effects of an individual bird's responses to wear-and-tear will be assessed in four ways: alterations in glucocorticoid function by measuring plasma hormone concentrations and glucocorticoid receptor dynamics; attenuation of sympathetic nervous system responses by measuring heart rate and heart rate variability; changes in behavior by measuring overall activity levels and responses to novel objects; and increases in homeostatic instability by measuring DNA damage as evidenced by changes in telomere length and cellular DNA fragmentation. The laboratory results will guide iterative alterations to the quantitative model, indicate new experimental designs based upon the updated quantitative model, and form revised predictions for subsequent laboratory tests of stress and wear-and-tear.