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ETHOLOGICALLY INFORMED DESIGN IN REPTILE RESEARCH |
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Neil Greenberg
Department of Ecology and Evolutionary Biology
University of Tennessee, Knoxville, Tennessee 37996
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published in
Health and Welfare of Captive Reptiles
C. Warwick, F.L. Frye, J.B. Murphy, editors
Chapman & Hall, London. 1994
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INTRODUCTION
There is a necessary relationship between research design, the welfare of research animals, and the validity of research data. This paper explores several dimensions of his relationship along with comments on the importance of ethologically informed design. Design, in the sense of a coherent program that guides a specific scientific undertaking, involves defining and selecting research variables and the methods that govern their manipulation and/or observation, measurement, and interpretation. Design both guides and is guided by the questions or problems an investigator wishes to address. To be ethologically informed, a design implicitly acknowledges four key processes or factors, each reflecting a different level of organization, but all profoundly interrelated in the causation of behavior: ecological, developmental, physiological, and evolutionary. These processes were identified in the earliest conceptual beginnings of ethology (Tinbergen, 1951) and continue to guide ethological thinking despite frequent fine-tuning of their respective domains or emphases (see Dewsbury, 1992). Questions about their role in behavior are characteristically answered by different modes of analysis, but when question, process, and mode of analysis are not carefully matched, much sterile controversy may be generated (Sherman, 1988). While these factors are related to each other, if only by being brought to bear on a common problem in understanding behavior, they are rarely considered conjointly because their research methods and historical traditions have served to isolate them. Such isolation and consequent specialization may be a necessary element in obtaining important disciplinary depth, but depth alone cannot bring research findings to full utility and may compromise validity. Research endeavors designed to accommodate the manner in which these variables affect the integration of internal and external stimuli in the causation of behavior (Lehrman, 1965) can be said to be ‘ethologically informed.’
ETHOLOGICALLY INFORMED DESIGN
A central tenet of ethologically informed design (EID) is that an intrusion into the natural functioning of any key process affecting behavior is likely to be reflected in the functioning of the others. While the importance of ethologically informed design is not limited to behavioral research, it is often most conspicuous there because of the sensitivity of most behavioral patterns to the integration of the many processes that underlie and converge in its expression. EID is obviously important to the validity of research findings, but it is also an element in the ethics of animal research because of the responsibility of researchers not to compromise animal welfare needlessly.
Validity refers to the effectiveness with which information corresponds to what we think it does, how free of bias it is, and the extent to which that information is related to the question being asked. For example, the most common, and perhaps unavoidable, challenge to the validity of our observations of animal behavior are misleading extrapolations from the areas with which we are most familiar, ourselves, (anthropomorphisms) or from misinterpretations based on our beliefs about other taxa (heteromorphisms).
A faulty EID –an ethologically uninformed design– may not destroy a research program but may distort its findings. For example, it should be obvious that the highly controlled captive conditions required for many physiological studies could lead to systematic neural or endocrine changes as animals attempt to compensate for the unnatural aspects of such environments. Changes in response to captivity may not challenge an animal’s survival or welfare in any obvious way and may in fact be highly reliable, that is, consistent and repeatable– but only in this restricted context. Such distortions are particularly difficult to detect because of the absence of adequate field studies to characterize the spontaneous variability of animals under natural conditions and thus their potential to adapt to a range of research conditions. An effective EID might recognize that consistency under controlled conditions may be a triumph of technique, but is not necessarily a virtue.
Less obvious but no less significant in impairing the validity of observations is ignorance of an animal’s developmental history. Experiences such as isolation, agonistic trauma, dietary deficiencies, or deprivation of key stimuli during sensitive periods can all have persevering effects (see Burghardt and Layne, this volume). At present, our information on how individual history affects life history variables of reptiles is scanty and we must take our cautions where we find them. Studies involving other taxa offer some insight. For example, in the laboratory, a well-meaning ethologist saw to it that newly hatched birds in the nest were well fed, only to see their parents reject them. Later, it was learned that in nature, hatchlings that do not beg for food are often discarded by their parents, presumably because they are sick or dead (Eibl-Eibesfeldt, 1970).
The effects of history on an animal need not, of course, be so dramatic or limited to a particular context. In a study on several vertebrate taxa including reptiles, it was found that occasional handling will affect weights of reproductive organs and fat stores (Meier et al., 1973). The degree of the effect depended upon the regularity and time of handling relative to photoperiod, and was presumably a result of transient mild stress. The likely effects of transient stress episodes, however, reminds us that it is reasonable to expect significant spontaneous variability in non-domesticated species. Animals in the field are certainly subject to occasional stress, and behavioral and physiological responses to stressors are so intimately involved with each other (see Hennessy and Levine, 1979) that there may be many ways that such episodes may even be necessary for the proper maturation of coping mechanisms.
Proximate and ultimate needs of animals
The welfare of animals utilized in research is typically characterized in terms of apparently dysfunctional behavior or expressions of distress or pain either in the course of captive maintenance or through a scientific procedure (reviewed by Morton, Burghardt, and Smith, 1990). Hierarchically organized ‘pain scales’ have been suggested (see Orlans, 1990), but as Bateson (1991a) points out, both the evolution as well as the functions of a subjective sense of pain is obscure. To assess animal well-being, both physiological and behavioral measures have been suggested and an enlightening debate has been engendered about their respective complexities and relative merits (summarized in part by Barnett and Hemsworth, 1990). Novak and Suomi (1988) identified four different approaches to defining psychological well-being: physical health, behavioral repertoire, reaction to stress, and responsiveness to environmental events. But some of these approaches, the authors point out, lead to contrary conclusions about how best to manage captive animals. Even within a specific approach such as reaction to stress, the great variability in the expression of distress between and within taxa makes generalizations overly dependent upon extrapolation from an alternative point of view. For example, when a presumed stressful stimulus (a live snake) is presented to squirrel monkeys, they will appear highly agitated and apparently stressed, but there is not necessarily an elevation in the stress hormone, cortisol, over what would result from mere novelty (Vogt, Coe, and Levine 1981). The behavioral and physiological perspectives have suggested various “objective” criteria for welfare as well as some sterile controversy, but the ethological appreciation for the complexities of adaptive change may help resolve differences. For example, well meaning efforts to maximize an animal’s welfare by minimizing an ‘objective’ measure of stress such as elevated circulating levels of adrenal hormone may deprive animals of opportunities to exercise and develop responses needed to deal with the normal variability in their environments. While not all needs are of comparable importance to welfare (Greenberg, 1992), we could argue that the ‘ultimate biological need’ of any animal is the realization of its maximum biological potential –its inclusive fitness, even where this involves the existence of a non-reproductive state.
Long-term expressions of stress may be most obvious in phenomena such as retarded development, impaired reproduction, and behavioral dysfunctions (see Christian, 1980), as well as more subtle phenomena such as accelerated aging and impaired immunocompetence, (reviewed by Johnson et al., 1992). Ultimately, our understanding of animal welfare hinges on our understanding of animal needs, but how can these be known? In von Uexkull’s words, “. . .animals construct nature for themselves according to their special needs” (1909/1985:234; emphasis mine).
Questions about animal needs and welfare necessarily call to mind human needs, often invoked to justify research — in particular the need to know and understand nature and the need for free, creative inquiry in pursuit of that goal. “Science for science’s sake” (echoing the nineteenth century battle cry by fighters for artistic freedom: “l’art pour l’art“) is believed indispensable for the atmosphere of free inquiry. But while creative inquiry is nurtured by freedom and may be an indispensible element of our confrontation with real or potentially dangerous ignorance of the natural world, there are almost universally acknowledged, if not universally practiced, constraints on even the freedom ‘to know.’
Laboratory and field
For experimentation, the scrutiny of presumably relevant variables while all other possible influences on dependent variables are held constant can lead to heroic efforts for control, if not uniformity, of all potential variables. However, the complexity of a animal’s interdependence with its natural habitat may dictate that meaningful study is possible only in nature or in a simulated habitat –even if this entails the sacrifice of fine control of relevant variables. Hopefully the sacrifice will be modest. An extreme example of faulty design was identified by Warwick (1990) who observed that restrictive environments that do not allow sufficient exercise may lead thereby to an array of behavioral and physical problems. It has long been appreciated that many debilitating dysfunctions in zoological parks are attributable to an incomplete understanding of the needs of captive animals (for example, Hediger, 1950, 1955). The concerns of zoological parks have undergone a marked transformation, however, such that modern zoos manifest much more interest in subtleties of welfare and behavior, particularly when they impact the reproductive effectiveness of endangered species (see Eisner, 1991). Special scrutiny has been afforded some of these problems in recent years with respect to environmental ‘enrichment.’ For example, Mahoney (1992) espouses a systematic investigation of the sensory capacities of captive animals and encourages accommodation of specific needs and aversions.
Frequently, even subtle aspects of a laboratory’s artificial setting may affect behavior in ways that underscore the importance of a previously unappreciated environmental influence on behavior. For example, the wavelength and intensity of ambient light may affect agonistic behavior of some lizards (Moehn, 1974), environmental scale may effect the timing activity patterns of captive anoles (DeLong, Greenberg, and Keaney, 1986), the interaction of light and temperature affects reproductive activity in male anoles (Licht, 1967), humidity may affect reproductive activity in female anoles (Summers, 1988, and see Stamps, 1976), and mild stressors may affect some but not all of the forms of tongue-flicking manifested by lizards (Greenberg, 1985).
Of course, artifactual or atypical behavior that may compromise laboratory work can also be introduced to the field and is sometimes is unavoidable. Frequently, only a subset of a population such as social subordinates or a particular sex or age class may be amenable to observation, and then, perhaps, only in specific parts of their environments such as an ecotonal boundary or periphery. Alternatively, Some individuals and not others may be aware of or sensitive to local disturbances associated with observations and adjust their behavior in response. Adult iguanas, for example, may change their home ranges or sleeping areas in apparent response to perturbations such as observing at night with a spotlight or the activity of constructing a blind 80 m away during the day; hatchlings may not disperse, but appear to increase the height of their sleeping perches where tall vegetation was available (Rodda et al., 1988).
For ethological studies, one effective compromise is to observe animals in the field and quantify temporal and spatial aspects of key life-history variables such as activity patterns, feeding habits, social activities, or the proportions of various daily activities. Once documented in the field, one may then observe the spontaneous expression of the same variables in an empirically-derived simulation of the natural environment in the laboratory. When comparable patterns of activity are expressed, the laboratory environment can then be gradually reduced in complexity as the animals are watched for indications that a removed variable was, in fact, important to the expression of the behavioral patterns of interest (Greenberg, 1978). A similar compromise may be attained by selectively simplifying the field setting by (for example) removal of obstructions, building of barriers to contain animals or facilitate quantification of their activities, or to hide observers from the view of animals. Such manipulations –“field experiments”– also include the introduction of controlled auditory, visual, or chemosensory stimuli associated with conspecifics, predators, or prey. For acquiring data that was once possible only under strictly controlled laboratory conditions, researchers in the field or using naturalistic habitats can now often employ attachable or even implantable transmitters that monitor variables ranging from activity levels to foraging or migratory movements to physiological variables. Occasionally, circumstances conspire to enable a coordinated field and laboratory studies. For example, Grassman and Hess were able to document sex differences in the seasonal variability of adrenal function in Cnemidophorus sexlineatus in the field (1992a), and then responses to acute stress in the laboratory (1992b).
While the common belief that we ‘observe in the field, experiment in the laboratory,’ is misleading, it reflects the fact that the context of inquiry, including the relative sense of control over variables, frequently affects the questions asked. Indeed, the world-views of field and laboratory workers often appear to reflect their respective experiences of nature, with diversity most apparent in the field and uniformity in the laboratory. It appears that both experiences should be incorporated into the training of all researchers.
Observation and experiment
As scientists, our knowledge of nature is grounded in observation. However not everything we might wish to know is comparably amenable to observation, requiring us to resort to artificial (and thus more-or-less trustworthy) extensions of our senses. The ‘logic of the lamppost’ describes the necessity to look for answers only where there is sufficient illumination to search. Suffering from either insufficient light or inadequate vision, many possible answers remain hidden in the deep shadows. Unfortunately, the experiments of nature –spontaneously occurring combinations of variables that we suspect may illuminate a particular problem– are rare. We must, as J.S. Mill put it, resort to ‘artificial’ experiments in order to add to nature’s experiments, those of our own devising. Because the essence of an experiment is the selection and selective control of relevant variables, we may extend Suzanne Langer’s (1957) famous dictum, “all real art is abstract,” to science. All useful research represents a selective emphasis of variables of interest –the abstraction of relevant details from a complex whole. Researchers such as ethologists trained to appreciate diversity as well as the common denominators in nature are alert to unique examples of what Mill identified as “nature’s experiments,” situations often created by combinations of environmental demands that selectively emphasize the variables of interest. It is precisely the selective representation of an attribute of interest that makes an animal model useful.
On a more ‘problem-focussed’ level, it is commonly accepted that while the findings of field studies possess more “external” validity –that is they are less vulnerable to distortion due to inadvertent behavior-altering artifacts– laboratory studies have greater “internal” validity because their findings are more reliable in the sense of being precise, sensitive, free of random errors, and replicable. Internal validity, however, implies a narrowness of applicability because the findings may be more-or-less limited to the precise conditions under which they were obtained. Commonly identified differences between field and laboratory include the less invasive nature of field work, the lack of standardization of field settings and its vulnerability to the vagaries of climate, the greater concern of field workers for natural populations rather than individuals, and the ‘need to know’ that motivates the investigation: field workers often appear more concerned with the animals than with the animal as a model (Orlans, 1988).
Context
It is a truism that the behavioral patterns seen in animals are profoundly affected by the context in which they are studied. Some of the most striking examples of the relevance of context-related stimuli are found in reptile learning. The well known difficulty in training many reptile species has engendered a sense of their resistance to learning (see Burghardt, 1977), however some lizards manifest a surprising capacity to learn in response to ecologically relevant stimuli (Brattstrom, 1978). Suboski (1992) suggests that the assumption that learning in reptiles is ‘impoverished’ is attributable in part to an inappropriate model of the learning process which neglects the importance of releasing stimuli. As Burghardt (1977) pointed out, expressions of behavioral plasticity such as learning are subject to gross misrepresentations by researchers if considered apart from ethological variables such as physiology or ecology. A relevant example emerged recently from a field study of the lizard Leiocephalus schreibersi, by Marcellini and Jenssen (1991). The researchers observed that one-trial acquisition of novel predator (human) avoidance behavior was manifested by 80% of the animals tested. Interestingly, the development of long flight distances developed more rapidly among females than males.
Many lizards habituate to human observers and appear to behave naturally (Amphibolurus, Lacerta muralis, Uta stansburiana, and some Anolis spp. see Sugerman, 1990) but some species appear resistant to habituation (Sugerman and Hacker, 1980). In the presence of an observer, captive Crotaphytus collaris will markedly reduce activity levels (Sugerman and Hacker, 1980), but the obtrusiveness of the observer is important. Even in the relatively insensitive anoles (Sugerman, 1990), an observer will increase the duration of tonic immobility (Edson and Gallup, 1972; see Gallup, 1974 for review), but probably in response to eye contact (Gallup, 1973). Further, the duration of the immobility responses manifested by A. carolinensis lizards in response to apparent predation (human handling) is significantly diminished in subsequent trials (McNight, Copperberg, and Ginter, 1978 and see Hennig, 1979).
While the ecological variables of predator-prey dynamics, geology, climate, and physical habitat are important in many obvious way, the subtlety of some of their interactions can be surprising: for example, slight differences in temperature can elicit alternative defensive responses to prospective predators (humans) in the lizard Anolis lineatopus (Rand, 1964). Approach distances (how near a potential predator may approach before triggering an escape response) are generally reduced at lower body temperatures (Rociia and Bergallo, 1990). Two other anoles, A. cristatellus and A. stratulus were observed to have significantly different approach distances in comparable thermal environments that appeared most likely correlated with the degree of crypsis they manifested: the more cryptic A. stratulus had a shorter approach distance (Heatwole, 1968). In Anolis carolinensis, the presumably defensive strategy of immobility when a prospective predator approaches is of significantly longer duration when foliage is nearby than when it is absent, but only in the early days of captivity (Hennig, 1979). The transfer of animals from the field to the laboratory is likely to result in altered behavior, but we cannot know without preliminary field work if the variations in behavior are within the range spontaneously manifested by the animal in nature.
CASE STUDIES: Ethologically integrated designs
A key goal of ethologically informed research is the reciprocal illumination that is provided by findings analyzed at different levels of analysis. Such studies represent points of articulation between ordinarily isolated disciplinary domains and by interconnecting different levels (Bateson, 1991b) they may become lynch-pins in the construction of an integrative overview of an organism and its place in nature. Ordinarily, such overviews are patched together from information derived from various species. In the following few pages I wish to briefly outline a few such areas in which theoretically fertile overviews are growing rapidly, primarily in lizards and frequently in Anolis carolinensis, the lizard with which I am most familiar.
Thermoregulation
Thermoregulation is perhaps the best studied of the physiological variables affecting reptile behavior. Since the work of Cowles and Bogert (1944 and see Bogert, 1959), few researchers would neglect the need for reptiles to regulate their body temperatures by selecting microhabitats of varying thermal qualities (see Heath, 1965; Lillywhite and Gatten, this volume). Between its central importance to physiological functioning and its amenability to documentation, investigations of the various thermal strategies of reptiles provide many points of articulation between different levels of organization and analysis. Ethologically integrative views of reptilian thermoregulation have focused on interlocking variables ranging from morphology (Pough, 1980) to mental capacities (Avery, 1976; Regal, 1980) and have helped dispel the ‘endothermocentric’ prejudice that uses birds and mammals as an evolutionary benchmark for success (Greenberg, 1976; Pough, 1980).
The richness of research into behavioral thermoregulation also provides us with examples of how validity compromised at one level can ramify through a cascade of subsequent research. For example, in adult male Anolis carolinensis, an appropriate body temperature is important to the responses of lizard adrenal to ACTH (Licht and Bradshaw 1969) and thus the entire ensemble of chronic stress effects on physiology and behavior. The attainment of a specific body temperature is known to affect the production and action of androgens (Pearson, Tsui, and Licht., 1976) and spermatogenesis in Anolis carolinensis (Licht 1971), as well as the hormonal state and reproductive status of other species (Licht, 1971; Garrick, 1974: Hutchison, Dowling, and Vinegar, 1966; Schwarzkopf and Shine, 1991; Daut and Andrews, 1993). Other physiological variables that involve thermoregulation and are also likely to affect behavior include diet, digestive state, and dehydration (Larson, 1961; Regal, 1966, 1980; Harwood, 1979), and even response to infection and disease (see Regal, 1980: Warwick, 1991). Thermal influences on muscle physiology (Licht, 1964a,b) might underlie the selection of specific defensive strategies at different temperatures (Rand, 1964 on Anolis, and see Hertz, Huey, and Nevo, 1982 on Agama and for a brief review), although “escape burst speed” and stamina documented in six genera of lizard by Bennett (1980) appear relatively independent of ambient temperature.
Progressive environmental effects on thermoregulation are most familiar in the phenomena of acclimation and acclimatization. These processes, indicative of an organism’s attempt to maintain homeostasis by compensating for an environmental change, exemplifies the complexity of interpreting research done in the laboratory versus that conducted in the field. When, in the laboratory, all conditions are held constant except for a single variable of interest (such as ambient temperature), the animal’s attempts to compensate are termed acclimation. In the more complex situation in the field (such as seasonal change), adaptive adjustments are termed acclimatization (Prosser, 1986). But in either event, different species can be expected to compensate at different rates (Art and Claussen, 1982) and in different ways. The periodically winter-active A. carolinensis apparently uses partial acclimation to cold as an overwintering strategy while the sympatric lizard, Cnemidophorus sexlineatus, hibernates rather than acclimate (Ragland, Wit, and Sellers, 1981). In the laboratory, following acclimation to an elevated temperature, Anolis carolinensis appears to be more heat-tolerant than those maintained at lower temperatures, but the temperature animals will spontaneously select (“mean preferred temperature”) is unaffected (Licht, 1968). Animals studied after acclimation in the laboratory are often assumed to be comparable to those acclimatized to the more complex environmental stimuli in the field, but this assumption may be unwarranted. Gatten, Echternacht, and Wilson, (1988), for example, observed oxygen consumption and lactate concentration at rest and during induced exercise at 20 C in two populations of green anoles, Anolis carolinensis: one acclimatized to various seasonal changes in the field and one acclimated to warm or cold conditions in the laboratory. In this study, resting oxygen consumption was unaffected by acclimation to warm or cold laboratory conditions, but did vary seasonally in the field animals. Oxygen consumption during exercise, however, showed significant differences between warm and cold acclimated lizards in the laboratory but was not affected by seasonal changes. Turtles are known to manifest an increase in their tolerance to abrupt temperature changes in response to increasing photoperiod and largely independent of seasonal temperature acclimation (Hutchison & Kosh, 1965).
Reproduction
Beyond mere survival, successful reproduction is the highest expression of the biological potential of individual animals. Because of the conservatism and complexity of reproductive processes, reproductive success can be regarded as an indication of well-being, and the environment in which this occurs may be regarded as at least adequate. Failure of captive or managed animals to produce successful progeny can, therefore, be regarded as a failure to fully understand their needs. For example, while adult Anolis spp. can be successfully maintained and will reproduce in the laboratory (Greenberg and Hake, 1990), rearing of progeny is notoriously difficult and rarely succeeds. Methods of facilitating reproduction in captive reptiles have thus rightly emphasized the convergence of external climatic, physical, and social stimuli on the internal processes leading to courtship, copulation, pregnancy, and parental behavioral patterns such as nest-site preparation, oviposition or parturition, and in some cases even brooding (see Crews and Garrick, 1980 and Carpenter, 1980).
Coordinated field and laboratory studies are often the only way to isolate and then reintegrate the subtly interdependent aspects of the complex reproductive process. For example, while opportunities for thermoregulatory basking would certainly be provided by most reptile ethologists, only recently have clues been forthcoming about the means by which the physiology of brooding females of various viviparous species affects thermal preferences in order to accelerate embryogenesis and decrease gestation time. The altered basking patterns of gravid females of several viviparous species was reviewed by Shine (1980), who noted that while there are several reproductive advantages, there are also distinct costs. In the laboratory and in the field, gravid female viviparous skinks, Eulamprus tympanum, will attain body temperatures comparable to those of males and non-gravid females, but will increase the duration of daily basking time. In the laboratory it was learned that the increased basking time results in accelerated gestation (Schwarzkopf and Shine, 1991).
Social dominance
In laboratory habitats, many lizards will manifest a continuum of degrees of territoriality (Hunsaker and Burrage, 1969). In the field, at least a few species of lizards also spontaneously shift their social organization from territoriality to a social dominance hierarchy in response to environmental changes (Evans, 1951 for Cteosaura pectinata, Norris, 1953 for Dipsosaurus dorsalis). Field data on social dominance in the well studied lizard, Anolis carolinensis is anecdotal at best, but in the laboratory, social dominance relationships are established with a rapidity suggestive of a well-established behavioral pattern (Greenberg, Chen, and Crews, 1984). Social dominance, in the conventional sense of one animal manifesting a priority of access to a limited resource over another, is seen in winners of aggressive interactions between males cohabiting a single enclosure. In this species, winners continue to perch at the highest site available and court females, expressing little more than occasional interest in the cohabiting loser. The loser, however, changes markedly: he becomes darker in body color, selects lower perch sites, is less active, and does not court –he has become a social subordinate. Such pairs often share food and water and maintain stable relationships for extended periods, suggesting a pattern well fixed in their behavioral repertoire. But observations from the lab, no matter how consistent, can do no more than suggest ecological hypotheses about the possible advantages accruing from the changes in subordinates: their lower posture, activity levels, darker color, and altered site selection.
Can we say anything as yet about the physiological substrate of altered social behavior? While the ecological aspects of the phenomenon in the laboratory are as yet elusive, the control and consistency of the laboratory facilitate investigations of the neurobiology and behavioral endocrinology. Earlier investigations established forebrain sites apparently responsible for integration of stimuli leading to the expression of specific social displays (reviewed in Greenberg, 1983, 1990), and brainstem nuclei controlling a key effector of the dewlap displays (Font, Greenberg, and Switzer, 1986; Font 1991). More recently, the darker body color of losers of fights provided a key to endocrine variables associated with social subordination and submissiveness. This phenomenon, well known in the laboratory (Greenberg and Noble, 1944) is also seen in the field (Medvin, 1990), and presents provocative possibilities because the hormones that affect the chromatophores are also associated with the physiological stress response and, at least in other taxa, have appear to act to suppress aggressiveness and facilitate the expression of social submissiveness (Leshner, 1978).
Many other reptiles are known to change color rapidly in response to environmental stimuli by means of chromatophore changes mediated by neural or neuroendocrine mechanisms (Cooper and Greenberg, 1992). The control of long-term color change, however, is in many cases is attributable to altered levels of circulating sex hormones as a result of seasonal or developmental changes, or chronic stress affecting adrenal cortical steroid hormone secretions. These longer-term changes can then alter the endocrine “tone” in which more rapid sympathetic neural / adrenomedullary changes are expressed in response to stimuli that may be potentially damaging, cryptic background-matching responses, or social signals which probably evolved from these more critical protective reflexes (Morris, 1956).
In Anolis carolinensis, body color responds rapidly to short-term or acute stressors as well as indicating long-term or chronic physiological adjustments in circulating hormone levels. This species represents a ‘natural experiment’ in that its dermal chromatophores are known to be free of sympathetic innervation (Kleinholz, 1938), leaving body color subject only to the influence of circulating hormones. Because these hormones, epinephrine (EPI), norepinephrine (NE) and melanotropin (melanocyte-stimulating hormone, MSH), also participate in the physiological acute stress response, body color provides useful clues about the internal state of the animal. MSH or relatively low levels of EPI causes darkening of melanophores, while NE or high levels of E will cause its lightening (Hadley and Goldman, 1969) (sympathetic nervous system responses in Figure 1). The hormone associated with the chronic stress response, the adrenal glucocorticoid, corticosterone (CS), does not usually affect color directly but can facilitate a key enzyme in the adrenal cortex thus increasing the rate of synthesis of EPI from NE, thereby also contributing to darker color (hypothalamic-pituitary-adrenal ‘cortex’ responses in Figure 1). Further, because one site on the body (the ‘eye spot’) is specifically devoid of hormone receptors that might balance or mask the effect of EPI, this species possesses a specific in situ ‘bioassay’ of adrenal ‘medullary’ (chromaffin cell) activation.
******************************* FIGURE 1*********************************
Laboratory studies of color change in A. carolinensis confirmed the sensitivity of chromatophores to stress-related hormones (Hadley and Goldman 1969). Later observations of the flux of stress-related hormones during aggressive interactions between recently introduced males and during a subsequent period of their cohabitation as dominant and subordinate, helped integrate chromatophore change with neural and endocrine activity and social organization (Greenberg, Chen, and Crews, 1984; Summers and Greenberg 1994).
In these latter studies, both body color and circulating levels of stress-related hormones were determined and found significantly altered in the predicted direction. For a further review of stress and physiology see Guillette, Rooney, and Cree (this volume).
Stress and reproduction.
The fundamental preliminary to all other needs is physiological homeostasis: the maintenance of an internal state that is stable within the range of variation an animal can experience before energetically demanding compensating responses are triggered. Within this range, sometimes termed ‘adaptive scope,’ animals can compensate with little or no energetic expenditure; beyond this range, a comprehensive physiological stress response is engaged which involves an ensemble of coordinated physiological and behavioral responses (Greenberg, 1990). Clearly, a failure to provide for one stress-sensitive variable can affect other variables as the stress response ramifies throughout the organism. Adaptive scope is influenced by experience such as acclimation or acclimatization, and the potential latent in the genome as it is expressed in a more-or-less environmentally sensitive or independent manner –the open or closed genetic programmes of Mayr (1974). In the absence of explicit information about the capacity of an organism to cope with a specific variable, we are obliged to limit potential challenges to a range within which it is known to be able to cope.
The stress response is an ensemble of coordinated autonomic and endocrine activities most elements of which are necessary parts of other essential systems (reviewed by Axelrod and Reisine, 1984; Johnson et al., 1992), serving both somatic and neurobehavioral (psychoactive) functions such as aggression and reproduction (Greenberg and Wingfield, 1987). The response is energetically demanding and if a limiting constituent is exhausted could lead to death, but without considering context and history it be difficult to interpret . For example, one of the key indications of stress is an increased levels of circulating adrenal corticosteroid, but at least in some taxa, this hormone can manifest bimodal effects stimulating feeding or activity such as running at low levels while decreasing both feeding and activity levels at elevated levels (reviewed by Leshner, 1978). Corticosteroids can even affect selective attention, impairing “perceptual restructuring” tasks after short term exposure and facilitating them after long-term exposure (Broverman, et al., 1974). The distinction between dangerous or potentially life-threatening stress and constructive uses of at least selected elements of the stress response is represented by the terms ‘distress’ and ‘eustress.’
Animals experiencing stress typically compensate by adopting a new behavioral or endocrine ‘tone’ –a new baseline state achieved by changing synthesis, secretion, binding, or receptiveness of target organs to stress-sensitive hormones. Examples would be the altered but stable endocrine profile of subordinate green anoles, Anolis carolinensis, (Greenberg, 1990), or lizards subjected to frequent handling (Meier et al., 1973). Because of the responsiveness of dermal chromatophores to stress-sensitive hormones, body color in many reptilian taxa can provide important clues about internal state and its regulation (Cooper and Greenberg, 1992, Greenberg, 1990, Hadley and Goldman, 1969).
Male sex hormones are associated with both the causes and consequences of dominant-subordinate relationships in many species (see, for example, Greenberg and Wingfield, 1987). Their natural elevation leads to aggressive interactions in which their circulating levels are reduced in losers. In Anolis carolinensis, winners of fights have a dramatic but transient androgen elevation while the levels in losers falls to about 50% normal in about a week (Greenberg and Crews, 1990). There is abundant evidence that increased adrenal activity in response to stressors can suppress reproductive endocrine activity and corresponding behavioral patterns. An adaptationist argument for the value of reduced androgen in subordinates is that when androgen facilitation of aggressive and reproductive behavior is absent, subordinates may avoid the potentially harmful responses of their dominant companion. The ensemble of stress responses is, however, so well integrated that
one could not determine the specific causal relationships of neural, endocrine, and behavioral phenomena without experimental intervention. For example, there is evidence that hormones of the pituitary-adrenal axis (ACTH and CS) exert independent effects on behavior, diminishing both aggressive and socially dominant behavior (reviewed by Leshner, 1978). In lizards, when CS levels of Anolis sagrei (Tokarz, 1987) or Uta stansburiana (DeNardo and Licht, 1993) are experimentally raised with hormone implants, display and attack behavior is markedly suppressed. In A. carolinensis, subordinate males spontaneously show elevated CS (Greenberg, Chen, and Crews, 1984) but unlike Uta stansburiana with experimentally elevated CS, they will not court females (Greenberg and Lumsden, 1990), possibly due to the inhibitory effect of the dominant male’s presence.
Whatever the precise nature of their interactions, the known psychoactive properties of gonadal and adrenal steroids converge in characterizing the endocrine profile of a non-aggressive, socially subordinate animal. Interestingly, the darker (brown) body color of subordinate A. carolinensis may be attributable to chromogenic effects of MSH, a pituitary peptide known to be co-released with ACTH; while these hormones have different effects on metabolism, their psychoactive effects are comparable. In some mammals, physical stress elevates both peptides, but psychological stress elevates only MSH (Sandman et al., 1973). Further, the effect of MSH at the receptor is affected by the hormones of the sympathetic-adrenal stress-response system: norepinephrine (Goldman and Hadley, 1970; Carter and Shuster, 1982) and epinephrine (Vaughan and Greenberg, 1987), presumably acting through a shared intracellular mechanisms. The modulation of the response to MSH by altered receptor dynamics might explain why, even in long-term (one month) subordinates showing consistently darker body color, circulating levels of MSH are not significantly higher (Greenberg, Dores, and Summers, unpublished data)
In A. carolinensis, castrated males will fight (if attacked) but rarely win territorial encounters with intact males. Although they lose encounters, they never develop the hormonal profile characteristic of stress seen in subordinate males: skin color remains green and CS levels do not go up (Greenberg, Chen, and Crews, 1984). In intact animals with reduced androgen subsequent to being defeated (Greenberg and Crews, 1990), behavior is much like that of castrates, but mild stress is indicated by darker body color.
RECOMMENDATIONS and CONCLUSIONS
Concerns of researchers for the welfare of their animal subjects have grown greatly in recent years. Whether or not this enhanced sensitivity is motivated by selfless concern for animal welfare or for producing the most useful or enduring research findings, we have become acutely, and I think, enduringly conscious of animal needs.
I venture an additional but modest proposal to facilitate the concerned researcher’s efforts. We may more fully accommodate animal needs if we had a clear sense of the consequences of animal use. To this end I suggest the use of a flexible checklists in which the elements of EID –variables relevant to environment, development, physiology, and evolution– are iterated in as much detail as possible for a given taxon. Recent developments in data management and access to data have the potential to make such an enterprise convenient and generally available.
The identification of a prospective research species to an EID database, for example, could convey researchers into a cascade of nested sublists providing progressively more detail on potentially relevant variables by reference to a central, universally accessible, regularly updated computer databank. By responding only to those domains of interest a researcher would rapidly approximate the ideal of an EID.
Prototype lists have already been generated to identify general experimental considerations in various reptiles (Warwick et al., 1992, Table 2), species-typical behavioral patterns that one might expect (Carpenter, 1977; Greene, 1988), and a detailed inventory of and social variables (McBride, 1976). Recommended parameters would then be provided where known from a database of ecological, physiological, and developmental needs or concerns for a particular species or for the next most closely related species, or even for species known to occupy comparable niches. In addition, conditions of captive maintenance known to be effective would be provided.
In conclusion, we cannot move quickly enough to secure the welfare of animals subject to the impact of human activities, nor can we be unrelenting in our efforts to facilitate the activities of researchers and ensure the utility of their labors. These closely related -often mutually dependent– concerns can be united and significantly enhanced by attempts to approximate the ideal of an ethologically informed design for a particular research project.
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Acknowledgements. I am deeply grateful for the sometimes spirited but always collegial and enriching comments and suggestions of Tom Jenssen and Gordon Burghardt; Clifford Warwick and an unidentified reviewer provided invaluable feedback.
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FIGURE 1
The effects of stress on a dermal chromatophore of Anolis carolinensis. Acute and chronic stressors are integrated to cause the release of hormones that interact with each other and converge in affecting the darkness of a chromatophore. ACTH, adrenocorticotropic hormone; CS, corticosterone; E, epinephrine; MSH, melanocyte stimulating hormone; NE, norepinephrine. CS can elevate the ratio of E to NE by facilitating a key enzyme within the adrenal gland. E stimulates receptors preferentially and then receptors resulting in pposing effects. (adapted from Greenberg and Crews, 1983)