HORMONES AND BEHAVIOR 18, l-1 1 (1984)

Social Status, Gonadal State, and the Adrenal Stress Response in the Lizard, Anolis carolhens&

NEIL GREENBERG, THOMAS CHEN, AND DAVID CREWS*

Department of Zoology, University of Tennessee, Knoxville, Tennessee 37996-0680, and *Institute of Reproductive Biology, Department of Zoology, University of Texas,

Austin, Texas 78712

Adult males of the small arboreal iguanid lizard, Anolis carolinensis, form social dominance hierarchies when placed in habitats with limited resources. Skin color changes occur during hierarchy formation, most conspicuously in subordinates, who appear darker (more brown) than dominants (more green). Because skin color in this species is under the control of hormones frequently associated with physiological stress, radioimmunoassay of plasma levels of the principal reptilian adrenal steroid, corticosterone, was performed. To examine the influence of gonadal androgen, known to inffuence the aggression that attends hierarchy formation, lizard pairs were constituted in which one or both members were castrated. Corticosterone levels of intact subordinates were signiiicantly elevated, whereas those of castrated subordinates or dominants showed levels comparable to those of isolates. No significant differences in spermatogenic stage could be detected between intact dominants or subordinates.

Many reptile species that are territorial in nature readily form dominance hierarchies in the laboratory or under certain natural conditions such as a sudden local food abundance (Evans, 1951, with Ctenosaura; Norris, 1953, with Dipsosaurus). In lizards, subordinate status is thought to be reinforced and maintained by dominant animals through agonistic inter- actions or by conspicuous morphological or behavioral indications of dominance to which subordinates are continually exposed (Harris, 1964; Greenberg and Crews, 1983).

The relationship of stress to the formation of social hierarchies was discussed by Rowe11 (1974), whose research on primates suggested that the stress to which subordinates are subject in captivity plays a major role in maintaining a hierarchy. Wittenberger (1981), however, reviewed evidence for the spontaneity and generality of social dominance, indicating that it is not merely, a stress-induced artifact of captivity. Studies of the relationship of social stress to adrenal physiology in many mammals and birds indicate that circulating levels of adrenal cortical stress hormones are lower in dominant than in subordinate individuals (e.g., Christian

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2 GREENBERG, CHEN, AND CREWS

and Davis, 1964; Louch and Higginbotham, 1967; Sassenrath, 1970; Myhre, Ursin, and Hanssen, 1981). Adrenal involvement in the physiological stress responses of lizards to psychological stimuli was suggested by the work of Callard, Chan, and Callard (1973, in Dipsosaurus and by Brackin (1978), who observed that in Cnemidophorus, enlarged adrenal glands were associated with low social status, The work reported here was undertaken to provide direct evidence for the association of relative social status with the adrenal stress response by measuring levels of circulating corticosterone, the principal interrenal (“adrenal cortical”) stress steroid of lizards (Sandor, Fazekas, and Robinson, 1976).

MATERIALS AND METHODS

Animals and maintenance regimen. The subjects are adult male green anole lizards, Anolis carolinensis, obtained from the Snake Farm, LaPlace, Louisiana.

Animals were placed in research habitats within 48 hr of capture in the field under conditions known to initiate and maintain gonadal recru- descence (Licht, 1971). The stimulatory regimen had a photic cycle of 14 hr light/l0 hr dark, with corresponding temperature fluctuations of 32-22°C. Temperature is known to be a significant variable in the responses of lizard adrenal to ACTH (Licht and Bradshaw, 1969), in spermatogenesis (Licht, 1971), and in the production and action of androgens (Pearson, Tsui, and Licht, 1976). Humidity varied inversely with temperatures from 70 to 90% RH. Food (3-week-old crickets) was available on a random schedule to maintain alertness. Handling was strictly minimized to eliminate this potential extra-experimental source of stress (Meier, Trobec, Hay- maker, MacGregar, and Russo, 1973).

Behavioral techniques. Prior to behavioral experimentation, 45 intact and 25 castrated adult males (minimum size, 61 mm snout to vent) were isolated for a minimum of 10 days in climate-controlled vivaria (22 x

22 x 22 cm). Isolated castrates were behaviorally stable and comparable to intacts along all dimensions recorded, including weight. After the period of isolation lizards were paired according to body size and weight in a neutral cage. The pairs were assigned to one of three experimental groups: 17 pairs of intact lizards (I x I), 9 pairs of castrates (C x C), and 7 pairs of one castrate and one intact each (I x C). A sham surgery group was not included on the basis of extensive work with castrates in several laboratories in which no effect on social behavior or reproductive physiology could be detected when castrations were incomplete or re- placement hormone was provided (see Crews, 1974; Crews, Traina, Wetzel, and Muller, 1978). Further, in preliminary tests, castrates were found to have corticosterone (CS) levels comparable to intacts (Greenberg and Chen, unpublished data).

The initial agonistic interactions were observed and documented on

STRESS AND DOMINANCE IN A LIZARD 3

videotape. The pairs were observed twice daily by instantaneous scan (time) sampling (Lehner, 1979, p. 122) until status relationships were stabilized (lo-30 days). In addition to direct observation, all behavior during a minimum of 14 hr of subsequent observations was documented by continuous time-lapse video recordings. Additionally, pairs were tested by observing their responses to a male or female intruder introduced to catalyze additional interactions.

Dominant status was ascertained by scoring wins, losses, and draws as determined by the performance of definitive acts such as an individual relinquishing a site or performing a subordinate, threat, or challenge display (Greenberg, 1977). Wins were defined as unopposed threat or challenge display or by other behavior causing a subordinate act in the partner, losses were defined by subordinate acts, and draws were recorded in the absence of winning or losing acts or when comparable behavior in both subjects occurred with no clear resolution. For quantification, losses were subtracted from wins and divided by the total number of encounters, including draws. The subject with the higher number was designated the dominant.

Body color. Skin color was assessed during the instantaneous scan observations for a minimum of 10 days before and 20 days following pairing. Scoring utilized a scale of seven mutually exclusive categories reflecting a progression from green (the color of sleeping animals or skin in tissue culture) to brown to green with an eyespot (reflecting “epinephrine reversal” ; Hadley and Goldman, 1969) to a final category of mottled green and brown with a prominent eyespot (Table 1). The significance of the change in the mean color score of subjects was determined by means of the Wilcoxon matched pair test (Siegel, 1956).

Radioimmunoassay of corticosterone. Chronic stress is indicated by increased ACTH secretion with consequent release of glucocorticoids from the interrenal tissue. As with birds, CS is the major adrenal steroid in lizards (Sandor et al., 1976). Findings from preliminary studies indicate that unstressed animals sacrificed within 10 set of capture will have

TABLE 1 Scale for Color Scores

Score Skin color pattern

All green Part green/part brown All brown Dark brown Dark brown with eyespot Green with eyespot Mottled green/brown

with eyespot

4 GREENBERG, CHEN, AND CREWS

reliably low or undetectable CS levels, thus minimizing potential handling- stress confounds (Greenberg, Chen, and Green, unpublished data).

Circulating CS levels were determined by radioimmunoassay (RIA) using antibody against corticosterone-3-BSA (No. 377) provided by Dr. Gordon D. Niswender from Colorado State University.

The principal buffer for the assay was 0.01 M phosphate-buffered saline solution containing 0.1% sodium azide and 0.1% bovine serum albumin (PBS-BSA), pH 7.4. The charcoal solution used to separate free from antibody-bound steroid was composed of 2.5% activated charcoal (Fisher) and 0.25% of Dextran T-70 (Pharmacia) in PBS-BSA buffer. The scintillation fluid consisted of 16 g of 2.5diphenyloxazole (PPO), 3.2 g of p-bis-(o- methylstyryl)-benzene (bis-MSB) in 4 liters of toluene (scintanalyzed, Fisher).

Blood samples were collected by rapid decapitation. Because levels of corticosterone may vary during the day (Chan and Callard, 1972), all blood samples were taken in the afternoon. Blood samples were spun at 1500 g for 10 min in a microfuge and the 50-200 ~1 serum obtained stored at -20°C until assayed. Since only small quantities of blood can be obtained and the nature of the steroids present in the blood was not known, two methods of preparing serum samples were employed in preliminary studies: (1) Serum was extracted with 2 ml dichloromethane and chromatographed in a Celite column using a gradient of isooctane- ethyl acetate as described by Siiteri (1975) to identify various steroids present in the blood. (2) The method of Klemm and Gupta (1975) for the RIA of plasma corticosterone in microliter quantities of blood was attempted. The second method was adopted. Briefly, this method utilizes less than 50 ~1 of plasma, heated at 60°C to destroy corticoid-binding globulins. Radiolabeled CS ([l ,2,6,7-3H]corticosterone, Amersham), y- globulin, and anti-corticosterone antibody are then added and incubated for 16 hr at 4°C. Unbound steroids were separated by ammonium sulfate precipitation and the radioactivity was measured in an Isocap 300 liquid scintillation counter. The efficiency’ for extraction was approximately 85%. The sensitivity of the assay is 16 pg. The intraassay and interassay variations were 10 and 13%, respectively. Concentrations of steroids in serum samples were calculated using the logit-log program of Rodbard and Lewald (1970). The significance of the difference between mean hormone levels of dominant and subordinate lizards was determined using the Mann-Whitney U procedure (Siegel, 1956).

Reproductive tract cytology and morphometric procedures. The re- productive tracts of males were removed, measured, and weighed. Sper- matogenic stage was assessed by the proportions of cell types (sper- matogonia, primary spermatocytes, spermatids, and spermatogonia) in the seminiferous tubules (Licht and Pearson, 1969). The renal sex segment,

STRESS AND ,DOMINANCE IN A LIZARD 5

a secondary sex characteristic, was also examined histologically to de- termine androgenic activity (Crews et al., 1978).

Surgical techniques. Castration was performed on subjects anesthetized in crushed ice. The testes were individually palpated through a s-mm incision on the lateral body wall, ligated, and removed according to Crews (1974).

RESULTS Circulating corticosterone. Plasma CS was significantly elevated (P =

< 0.05) in subordinates (z = 18.8 &ml) relative to dominants (x = 4.9 nglml) (Fig. 1). The CS levels of dominants were comparable to those of isolates (x = 1.5-3.0 rig/ml, depending on time of day; n = 28; Greenberg, Green, and Chen, unpublished data). In pairs in which one subject was castrated, the intact always became dominant. The subordinate individuals in I x C pairs and in C x C pairs did not show significantly elevated circulating CS levels relative to dominants, although variability was increased (Fig. 1).

Reproductive cytology of intact dominant and subordinate males. Fourteen pairs of lizards were suitable for relative spermatogenic stage assessment. In six of these, subordinates were less than two stages more

30 F

1 IX1 1 XC cxc

FIG. 1. Serum corticosterone (CS) levels in socially dominant and subordinate male An& carolinensis. I x I, Matched pairs consisf of two intact lizards; I x C, pairs cansisting of one intact and one castrate; C x C, pairs consisting of two castrates. Horizontal line represents mean; vertical bars represent 2 standard errors. The elevation in intact subordinate CS levels is significant at P = < 0.05 (Mann-Whitney U test).

6 GREENBERG, CHEN, AND CREWS

advanced spermatogenically than their dominant cagemates. In eight pairs, dominants were more advanced spermatogenically. In six of these pairs the dominant was two or less stages advanced, and in two pairs the dominant was at least five stages more advanced.

Body color of intact and castrate dominant and subordinate males. In all pairs, lizards that were socially dominant exhibited a skin color comparable to their color as isolates, although variability was increased in castrated relati’ve to intact dominants. Subordinates, on the other hand, had significantly darker skin if intact but showed no notable change from their isolation color score if castrated (Fig. 2). The eyespot, a positive indication of the presence of epinephrine (Hadley and Goldman, 1969), was seen only during or immediately following aggressive interactions.

DISCUSSION

A. carolinensis provides an excellent model for the examination of hypotheses related to stress physiology and other autonomic aspects of aggression and status. The finding of elevated CS in subordinate lizards is consistent with findings in other taxa (e.g., Louch and Higginbotham, 1967, for mice; Myhre et al., 1981, for ptarmigan).

It is significant that in all pairs in which a stable dominance relationship was established, the intact subordinate individuals had a relatively darker skin color. Subordinates also frequently adopt perch sites in which a darker skin color would be cryptic (Greenberg, unpublished data). Color changes in A. carolinensis during the establishment of dominance hier- archies are of particular interest in this regard because they may serve as in situ bioassays for stress-related neuroendocrine phenomena. In this

+ 1.0

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f -0.6

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III Ix1 I x c cx c

FIG. 2. Mean changes in body color associated with social status after pairing. Positive changes represent darker color. Changes are based upon’scores assigned for color before and after pairing (see Table 1). Horizontal lines represent mean changes and vertical bars represent 2 standard errors. The elevated scores (skin darkening) of I x I subordinates is significant at P = < 0.01 (Wilcoxon matched pair test).

STRESS AND DOMINANCE IN A LIZARD 7

species body color is, perhaps uniquely, under the exclusive control of circulating hormones associated with stress physiology (reviewed by Greenberg and Crews, 1983). In A. caroline~~.sis, corticosterune may affect body color indirectly due to its facilitation of the key methylating enzyme in the biosynthetic pathway of epinephrine (Wurtman and Axelrod, 1965; Wurtman, Axelrod, and Tramezzani, 1967), known to darken skin color in this species (Hadley and Goldman, 1969). Skin color is also influenced by the hormone of the pituitary pars intermedia, melanocyte- stimulating hormone (MSH). Interestingly, A, carolinensis will release MSH from a subcutaneous pars intermedia graft when stressed (Meurling, Klefbohm, and Larsson, 1974). In mammals, MSH as well as ACTH release is significantly influenced by hypothalamic corticotropin releasing factor (CRF) (Proulx-Ferland, Labrie, Dumont, and Cote, 1982). If a similar mechanism occurs in A. carolinensis and the ACTH or consequent adrenocortical response to CRF declines with time while that of MSH remains, the relatively modest CS response in long-term subordinates would avoid the deleterious physiological responses to continuing stress while maintaining the adaptive MSH-induced darker body color. Persistent high circulating MSH levels can also lead to morphological darkening (see Bagnara and Hadley, 1973), but since most chronically dark A. carolinensis can lighten rapidly in certain situations, any morphological changes would be most likely manifest as an increased responsiveness to physiological stimulation.

It is likely that the initial CS response to the change in social status experienced by subordinates is more extreme than that documented here in long-term cohabitants (Bronson, 1973; Myhre et al., 1981). In mice, the plasma CS response sensitively reflects the intensity of psychological stimulation (Hermessy and Levine, 1978) more accurately than does ACT (Hennessy, Heyback, Vernikos, and Levine, 1979). Leshner and Polite (1979) have suggested that while elevated ACTH is independently re- sponsible for diminished aggression in mice, elevated CS is the important element in submissiveness. Interestingly, the difference in dominance scores shown between subordinate castrates and dominants reflects more a lack of aggression than an expression of submissiveness. While this study does not address the issue of the relative importance of MSH, ACTH, or CS in the control of either subordinate social status or darker body color, it does clearly indicate adrenal activation. The precise lationship of these hormones to social behavior is the subject of continu investigation.

Although subordinate lizards are reproductively inactive, reproductive condition is not obviously related to social rank in A. carolinensis as indicated by reproductive tract cytology. Indeed, reproductive physiology in general is less clearly related to social rank than is adrenal activation in several species. In mice, decreases in seminal vesicle weights were

8 GREENBERG, CHEN, AND CREWS

correlated with exposures to trained fighters (Bronson and Eleftheriou, 1964) but in sparrows, no correlation between dominance and testosterone could be detected (Rohwers and Wingfield, 1981). It might be highly adaptive for subordinates, while behaviorally nonreproductive to be physiologically capable of rapidly replacing a dominant defeated in combat or lost to a predator.

In A. Caroline&s, castration eliminates reproductive behavior (Crews, 1979) but aggression may remain intact for at least 2 weeks, probably due to differences in neural thresholds for the effect of androgen (see Crews and Greenberg, 1981). Alternatively, aggression may not be under androgen control directly, but occur in response to contextual stimuli. For example, castrates need to be returned to their home cages for territorial behavior to appear intact (see Crews, 1974). In the present study, all castrates engaged in aggressive interactions when paired but ultimately became subordinate if cage cohabitants were intact. The behavior of castrates that became dominant in the C x C group was comparable to that of intact dominants. In two subjects not included in the present analysis, all indications of social dominance were present although cas- tration was found to be incomplete.

The absence of a CS elevation in castrated subordinates indicates reciprocal influences between the adrenal and gonadal axes. Although Kitay (1968) suggested that reduced adrenal CS secretion after gonadectomy in rats was probably attributable to a sex steroid action on the hypothalamic- pituitary-adrenal axis, this has been little studied. Chronic stress can delay sexual development in rodents, probably by means of an ACTH- induced reduction in gonadotropin (LH) secretion. This is mediated by the adrenal gland in rats but not mice (see Mann, Jackson, and Blank, 1982), and its clarification in reptiles awaits future work. Testosterone appears to synergize with ACTH in action on CS secretion in hypophy- sectomized rats (Colby, 1978), but behavioral as well as physiological effects must be considered. Gonadal steroids could alter selective attention (Oades, 1979) or persistence of attention (Andrew, 1978), as well as altering sensory function directly (Gandelman, 1983) in ways that might ameliorate the effects of psychological stressors. If reduced androgen provides a measure of relief from the well-known deleterious effects of chronic stress, an added dimension to our understanding of coping re- sponses may well emerge.

ACKNOWLEDGMENTS We wish to express our gratitude for the expertise and commitment of senior research

technician Helen Green and student laboratory assistant Laura Hake. Editorial suggestions of Alicia Berry Greenberg and an unidentified reviewer are gratefully acknowledged. This work was supported by NIH BRSG RR-07088 and a Schweppe Research and Education Fund Award to N.G. and NICHHD 16687 and NIMHRSDA 00135 to D.G.

STRESS AND DOMINANCE IN A LIZARD

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