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Male Morphs in Tree Lizards Have Different Testosterone Responses to Elevated Levels of Corticosterone
General and Comparative Endocrinology 107, 273–279 (1997) Article No. GC976923
Male Morphs in Tree Lizards Have Different Testosterone Responses to Elevated Levels of Corticosterone Rosemary Knapp1 and Michael C. Moore Department of Zoology, Arizona State University, Tempe, Arizona 85287-1501 Accepted April 16, 1997
Changes in circulating glucocorticoid and androgen levels mediate agonistic behaviors in many vertebrates. Individual variation in the magnitude of the glucocorticoid response to stressful stimuli, the negative effects of elevated glucocorticoid levels on androgen levels, or both could mediate individual differences in subsequent agonistic behavior. In a series of previous studies, we found that both alternative male reproductive morphs in the tree lizard, Urosaurus ornatus, can exhibit elevated levels of plasma corticosterone following male–male encounters, but that the territorial morph appears less likely to exhibit coincident decreases in plasma testosterone. Two studies tested the hypothesis that the two morphs differ in the degree to which testosterone levels are influenced by elevated corticosterone levels. In the first study, physically restraining males elicited endogenous elevations of circulating corticosterone levels. Testosterone levels were significantly negatively correlated with corticosterone levels in the nonterritorial morph, but there was no correlation between levels of the two steroids in territorial males. In the second study, corticosterone levels were artificially elevated in freeliving male tree lizards using a noninvasive dermal patch. This exogenous elevation of corticosterone significantly depressed testosterone levels in both morphs, but it produced a significantly greater depression in the nonterritorial morph. Nonterritorial males appear to be more sensitive than territorial males to the testosteronesuppressing effects of elevated circulating levels of 1
Present address: Section of Neurobiology and Behavior, Cornell University, Mudd Hall, Ithaca, NY 14853. 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
corticosterone. This difference between the morphs in the effects of a stress hormone on the reproductive axis may be a fundamental part of the mechanism (1) underlying behavioral tactic switching within the nonterritorial morph or (2) contributing to behavioral differences between the morphs. r 1997 Academic Press In many vertebrates, there is a negative effect of glucocorticoids on androgen production. This provides one route by which stress can potentially suppress the reproductive axis. However, the magnitude of the effect of glucocorticoids on androgens often varies considerably among individuals. Variation in these hormone responses may arise from genetic differences (Shire, 1979; Brown and Nestor, 1973; Gentsch et al., 1982, 1988; Satterlee and Johnson, 1988; Brush et al., 1991) or from different social experiences (Sapolsky, 1985; Knapp and Moore, 1996). Since both glucocorticoids and androgens are known to influence agonistic behavior (Leshner, 1980; DeNardo and Licht, 1993), individual variation in the magnitude of the negative effects of glucocorticoids on androgen levels could contribute to individual variation in agonistic behavior. Our recent work suggests the possibility that such a mechanism underlies behavioral variation between the two alternative male reproductive morphs of the tree lizard, Urosaurus ornatus. The two morphs differ in their likelihood to escalate agonistic encounters and in their territorial behavior (Hover, 1982, 1985; Thompson and Moore, 1991b, 1992). This behavioral variation
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is correlated with color variation in the dewlap (throat fan), a structure used in social communication. Males with orange dewlaps containing a central bluish-green spot (‘‘orange–blue males’’) are territorial and very likely to escalate male–male interactions. Males with orange dewlaps but lacking a central bluish-green spot (‘‘orange males’’) are nonterritorial and less likely to escalate male–male interactions. In a series of previous studies, we found that both morphs can exhibit elevated plasma levels of corticosterone 1 day following male–male encounters, but that the territorial morph appears less likely to exhibit coincident decreases in plasma testosterone (Knapp and Moore, 1995, 1996). These results suggested the hypothesis that the morphs may differ in the degree to which the hypothalamic–pituitary–gonadal (HPG) axis is suppressed by activation of the hypothalamic– pituitary–adrenal (HPA) axis. We therefore tested this hypothesis in two ways. First, we examined testosterone levels following endogenous elevation of corticosterone levels in response to restraint stress. Second, we examined the testosterone response of the two morphs to exogenous elevation of corticosterone levels. The hypothesis predicts that testosterone levels would be influenced by elevations in corticosterone to a greater extent in orange males compared with orange– blue males. The results of the two studies reported here support this prediction and, in addition, suggest that this morph difference is expressed only when corticosterone levels are elevated.
ENDOGENOUS CORTICOSTERONE ELEVATION Methods Field site. The study was conducted in Tonto National Forest within an approximately 5-km radius of the intersection of Sugarloaf Mountain–Sycamore Creek Road with Arizona State Highway 87. This area has been used regularly by our laboratory since 1986 (see Thompson and Moore, 1991a, for a full description of the site). This study was conducted between June 2 and August 6, 1988, during the active breeding season (April–August). Females may produce one to three clutches of eggs during a breeding season, and recep-
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Knapp and Moore
tive females are present throughout all but the very end of the breeding season (Tinkle and Dunham, 1983). Animal handling. Glucocorticoid levels increase in response to physical restraint in many vertebrates (e.g., Moore et al., 1991; Wingfield et al., 1995). To induce endogenous increases in corticosterone levels, male tree lizards (n 5 16 for each morph) were captured by noosing and then transferred to cloth bags for 10 or 30 min (see Moore et al., 1991). Two sampling time points were used to produce a range of corticosterone levels. A blood sample was then collected from the orbital sinus using heparinized microcapillary tubes and held on ice until return to the laboratory (within 4 hr). There the samples were centrifuged for approximately 5 min. Plasma was collected and stored at 220° until assayed. Hormone assays. Steroid hormone levels were determined by radioimmunoassay following chromatographic separation (Moore, 1986; Knapp and Moore, 1995). Prior to chromatographic separation, plasma samples were extracted in diethyl ether and then resuspended in 10% ethyl acetate in isooctane. Testosterone and corticosterone were then separated from each other and from interfering neutral lipids using celite minicolumns [a 3 g celite:1 ml ethylene glycol: propanediol mixture (1:1) over 3 g celite:1 ml water ‘‘glycol trap’’]. Each steroid was collected as a separate fraction by stepwise elution using increasing concentrations of ethyl acetate in isooctane. Fractions were dried down under nitrogen and then resuspended in buffer. Aliquots of each resuspended fraction were assayed in duplicate for each sample using radioimmunoassay. Values were corrected for individual sample recovery calculated from recovery of small amounts of radiolabeled testosterone and corticosterone that were added to each sample prior to ether extraction. Plasma samples were assayed in two assays. Intra-assay variation was 7.0% for both testosterone and corticosterone; interassay variation was 7.5% for testosterone and 11.8% for corticosterone.
Results Data were analyzed by regressing testosterone levels on corticosterone levels. Such an analysis factors out potential effects of time of the breeding season on hormone levels (Moore and Thompson, 1990). The relationship between corticosterone and testosterone differed significantly between male morphs (Fig. 1).
Testosterone Responses in Lizards
FIG. 1. Morph differences in the relationship between plasma testosterone and corticosterone levels in response to restraint stress. Lines illustrate least squares regressions and their 95% confidence intervals.
The slopes of the regressions for the two morphs differed significantly (t 5 2.73, df 5 30, P , 0.02). The slope of the log–log regression of testosterone on corticosterone was negative (20.63, r 2 5 0.70) and significantly different from zero for orange males (t 5 25.68, df 5 15, P , 0.01), but for orange–blue males was not significantly different from zero (slope 5 0.01, r 2 5 0.00; t 5 0.05, df 5 15, P 5 0.96). However, mean levels of each hormone did not differ significantly between the morphs [corticosterone (mean 6 SE): orange, 10.9 6 2.6 ng/ml; orange–blue, 16.6 6 3.8; t 5 1.25, df 5 30, P 5 0.22; testosterone: orange, 23.7 6 2.8 ng/ml; orange–blue, 23.1 6 3.6; t 5 0.12, df 5 30, P 5 0.91].
EXOGENOUS CORTICOSTERONE ELEVATION Methods The data presented in this section are part of a larger study examining multiple effects of corticosterone on male morph differences in physiology and behavior. Here we describe the portion of this larger study that was designed to test the hypothesis that the two male morphs differ in the degree of testosterone suppression following exogenous elevation of corticosterone. Animal handling. This study had two stages. First, males were captured, given an experimental manipula-
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tion, and released at the site of capture (Day 1). On the following day (Day 2), males were recaptured and a blood sample was collected. This study was conducted between May 18 and July 11, 1995, at the same field site as the study described above. Males of both morphs were captured by noosing as they were encountered on the study site on Day 1. Only males with tails that were at least 75% of their full length were included in the study because tail loss affects social interaction and home range size in other lizard species (Fox et al., 1990; Salvador et al., 1995). Following capture, males were scored for dewlap color, toe clipped for permanent identification, measured for snout–vent length (nearest mm), and weighed to the nearest 0.1 g on a portable electronic balance. Each male was also given a unique paint mark on the base of the tail to facilitate resighting on Day 2. Males were then given a dermal patch containing 4.5 µl of either (a) a solution of 3 µg corticosterone/µl sesame oil (total, 13.5 µg corticosterone; ‘‘corticosterone patch’’) or (b) sesame oil only (‘‘oil patch’’) (n 5 5–11). These patches were constructed from small pieces of adhesive bandage and vinyl electrical tape that were secured to the males’ backs by means of a transparent dressing (Johnson & Johnson Bioclusive; see Knapp and Moore, 1997, for a detailed description of the patch). Males were assigned alternately to one of the two patch treatment groups in the order they were captured. Total handling time was always less than 12 min, with almost all animals being handled for only about 5 min. Males were released at the site of capture and the location was marked with flagging and noted on a sketch map of the area. The following day (Day 2), males were resighted by searching the general vicinity of the marking site for up to 20 min. Once males were resighted, they were exposed either to an orange–blue intruder male tethered to a pole or only to the pole for a period up to 5 min. [This manipulation of exposing experimental males to an intruder male or a pole was relevant for other aspects of the larger study and is reported here only to accurately describe animal handling. Corticosterone and testosterone levels have been shown not to change within 60 min of male–male interaction in this species (Thompson and Moore, 1992; Knapp and Moore, 1995). Male–male interaction also had no significant effect on hormone levels in the present data set (t
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tests with Bonferroni correction for multiple comparisons, data not shown).] Following the behavioral test, males were again captured by noosing. Blood samples were collected from the orbital sinus using heparinized microcapillary tubes (completed within 1 min of capture) and held on ice until return to the laboratory (within 4 hr). There they were centrifuged for approximately 5 min. Plasma was collected and stored at 220° until assayed. Blood samples were also collected from an additional group of unmarked males of both morphs (‘‘no patch,’’ n 5 16–18). These males had not been handled the day before but were otherwise treated exactly as were males with patches and served as controls for possible effects of handling on hormone levels. These males were sampled on the same days that blood samples were collected from males with patches to control for abiotic factors such as weather that could potentially influence hormone levels. Hormone assays. Plasma samples were assayed for corticosterone and testosterone as described above. For this study, all samples were measured in a single assay in which intra-assay variation was 9.7% for testosterone and 5.9% for corticosterone. Statistical analyses. Hormone data were logarithmically transformed to meet assumptions of normality required for analysis of variance. Testosterone and corticosterone levels in male tree lizards are known to vary across the breeding season (Moore and Thompson, 1990). Therefore, preliminary ANCOVAs were performed with numerical day of the year (‘‘season’’) as a covariate to determine whether this was also the case in the present data set. Season was a statistically significant covariate for testosterone (F(1, 59) 5 4.56, P 5 0.037), but not corticosterone (F(1, 59) 5 0.13, P 5 0.721). Corticosterone data are presented as backtransformed means and standard errors from the analysis of variance (Fig. 2A). Testosterone data are presented as back-transformed adjusted means and standard errors from the ANCOVA to show the influence of treatment after the effect of season has been removed (Fig. 2B). Preliminary analyses also indicated that hormone levels of the two control groups did not differ for either morph (ANOVA and ANCOVA, both P . 0.13). The two control groups for each morph were therefore combined for further analyses. We present the data for the individual control groups (no patch
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Knapp and Moore
FIG. 2. Plasma corticosterone (A) and testosterone levels (B) of free-living males from the exogenous corticosterone experiment. Data are presented as back-transformed means (mean 6 SE) from analyses of variance. Hormone levels for the two control groups (no patch and oil patch; left side of figure) did not differ significantly for either morph and were therefore combined (controls) for further analysis (see text). Means with different letters for each hormone (right side of figure) are significantly different from each other, P , 0.05. Sample sizes are given between the graphs. k, orange males; b, orange–blue males.
and oil patch) in Fig. 2 for the reader’s information. Post hoc Student Newman–Kuels (SNK) tests were performed following ANOVAs to determine significant pairwise differences.
Results As expected, patch treatment had a significant effect on plasma corticosterone levels in both morphs (Table 1; Fig. 2A). Corticosterone levels of orange and orange– blue males receiving corticosterone patches were significantly elevated compared with controls (SNK tests: both P , 0.001). Corticosterone levels of the orange and orange–blue controls did not differ significantly from each other (SNK tests: P . 0.20). Corticosterone levels of orange and orange–blue males receiving corticosterone patches also did not differ (SNK test: P . 0.50). The magnitude of corticosterone elevation produced by the corticosterone patches was in the range of levels that tree lizards are capable of produc-
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Testosterone Responses in Lizards
ing endogenously under stress (Fig. 1 and Moore et al., 1991). Corticosterone patch treatment also significantly affected plasma testosterone levels (Table 1; Fig. 2B). Males of both morphs receiving corticosterone patches had significantly lower testosterone levels than controls (SNK tests: both P , 0.001). However, the morphs differed in the degree of testosterone suppression resulting from corticosterone patches; orange males had significantly lower testosterone levels than did orange–blue males (SNK test: P , 0.025). Testosterone levels of orange and orange–blue controls did not differ significantly (SNK test: P . 0.50). At the low corticosterone levels exhibited by the two control groups (no patch and oil patch), there was no significant relationship between circulating levels of corticosterone and testosterone for either morph (Fig. 3); the slopes of the log–log regressions of testosterone on corticosterone were not significantly different from zero (orange: 20.12, r 2 5 0.013; orange–blue: 5 0.11, r 2 5 0.017; both P $ 0.50).
DISCUSSION The two behavioral morphs of male tree lizards differ in the degree to which circulating testosterone levels are affected by elevation of plasma corticosterone. Testosterone levels in the less aggressive, nonter-
TABLE 1 Analysis of Variance Results for Plasma Corticosterone and Testosterone Levels for Exogenous Corticosterone Study Source Corticosterone Treatment Morph Treatment 3 morph Error Testosterone Season (covariate) Treatment Morph Treatment 3 morph Error
df
MSS
F
P
1 1 1 62
12.85 0.06 0.00 0.08
156.77 0.78 0.00
0.000 0.382 0.962
1 1 1 1 61
0.32 3.34 0.48 0.25 0.08
3.92 40.50 5.82 3.03
0.052 0.000 0.019 0.087
Note. Data were logarithmically transformed prior to analysis to meet the assumptions of analysis of variance. Season refers to the numerical day of the year.
FIG. 3. Plasma testosterone levels exhibit no significant relationship to corticosterone levels in control males (no patch and oil patch of Fig. 2) from the exogenous corticosterone study. Lines illustrate least-squares regressions and their 95% confidence intervals.
ritorial orange males were suppressed to a greater degree than were testosterone levels in the more aggressive, territorial orange–blue males. This morph difference in plasma testosterone levels occurred despite corticosterone levels being elevated equivalently in the two morphs by the experimental manipulation in each study. Further characterization of morph differences in sensitivity of testosterone levels to elevations in corticosterone requires a dose–response study. Such a component could not be included in the exogenous corticosterone study due to logistic constraints, but will be important for fully characterizing the morph difference described here. Glucocorticoids can modulate HPG axis activity at the level of the hypothalamus, pituitary, or testes in other vertebrates (Rivier and Rivest, 1991). Direct inhibitory effects of glucocorticoids at the level of the testes have been described in several species (Bambino and Hsueh, 1981; Cumming et al., 1983; Sapolsky, 1985; Orr and Mann, 1992). The site(s) of action of the suppressive effects of elevated corticosterone levels on male tree lizards, and whether this morph difference is genetic, remains to be determined. The morph difference in response of testosterone levels to corticosterone appears to exist only when corticosterone levels are elevated (compare Figs. 1 and 3). Previous studies of male tree lizards have also failed to find differences in the testosterone–corticosterone relationship for the two morphs when hormone levels are basal (Moore and Thompson, 1990; Thompson and Moore, 1992; Knapp and Moore, 1996). These
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results are reminiscent of findings that strain and experience differences in glucocorticoid levels in laboratory animals are evident only in response to moderate stressors and not under basal conditions (Gentsch et al., 1988; Meaney et al., 1991). Such differences among groups in laboratory animals may be mediated by different densities of glucocorticoid (Type II) corticosteroid receptors in certain brain regions (Meaney et al., 1991). At basal circulating levels of glucocorticoids, mineralocorticoid (Type I) receptors have a high rate of occupancy and are thus relatively insensitive to changes in corticosterone levels. Glucocorticoid (Type II) receptors, which have low occupancy rates at basal corticosteroid levels but high occupancy rates at elevated corticosteroid levels, are more responsive to elevations in circulating corticosterone. In addition, effects of corticosteroids on targets outside the HPA axis appear to be primarily mediated by glucocorticoid (Type II) receptors whereas targets within the HPA axis appear to be primarily mediated by association of corticosteroids with mineralocorticoid (Type I) receptors (Akana et al., 1992). Whether lizards possess two types of corticosteroid receptors as do mammals remains to be determined, although it seems likely given that they do secrete the mineralocorticoid aldosterone (Hadley, 1992). If tree lizards are found to possess two types of corticosteroid receptors, our data lead us to predict that orange males would possess more Type II receptors than orange–blue males in brain areas mediating HPA–HPG interactions. The greater suppression of testosterone levels in nonterritorial orange males in response to elevations in corticosterone is consistent with a hypothesized hormonal mechanism contributing to switching of reproductive tactics exhibited by these nonterritorial males. We have recently documented year-to-year variation in behavioral tactics of orange males (Knapp, 1996). When environmental conditions are relatively benign and social interactions are not particularly intense, orange males appear to adopt a sedentary satellite tactic. Under more stressful conditions that could induce elevations in circulating corticosterone, orange males apparently act as predominantly non-sitefaithful rovers. In contrast, orange–blue males exhibit the same reproductive tactic (territoriality) under all conditions. Based on the documented behavioral roles of corticosterone and testosterone, we hypothesize that
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Knapp and Moore
the elevation of corticosterone levels for orange males and the coincident decrease in testosterone levels in response to prevailing social and/or environmental stressors are part of the neuroendocrine mechanism influencing which reproductive tactic is exhibited. For orange–blue males, resistance to elevating corticosterone levels following male–male interactions (a social stressor) (Knapp and Moore, 1996), reduced testosterone suppression following elevations in corticosterone levels in response to physical or environmental stressors (this study; Knapp, 1996), or both may help to maintain aggressiveness at the level required for successful territorial maintenance (see also Moore et al., in review). Taken together our data support the speculation that a difference in the HPA axis may be an important component of the mechanism mediating the different behavioral responses of the two morphs to their social and physical environment.
ACKNOWLEDGMENTS We thank Alan Pearlman for collecting the blood samples for the endogenous corticosterone study and Diana K. Hews, Eva L. Lacy, Danika L. Painter, and Sarah K. Woodley for assistance in several aspects of the exogenous corticosterone study. This work was conducted under guidelines established by the ASU Animal Care and Use Committee and appropriate permits from Arizona Game and Fish and the United States Forest Service. Financial assistance for this work was provided by an NIMH predoctoral National Research Service Award to R.K. and NIMH MH48564 to M.C.M.
REFERENCES Akana, S. F., Scribner, K. A., Bradbury, M. J., Strack, A. M., Walker, C.-D., and Dallman, M. F. (1992). Feedback sensitivity of the rat hypothalamo–pituitary–adrenal axis and its capacity to adjust to exogenous corticosterone. Endocrinology 131, 585–594. Bambino, T. H., and Hsueh, A. J. W. (1981). Direct inhibitory effect of glucocorticoids upon testicular luteinizing hormone receptor and steroidogenesis in vivo and in vitro. Endocrinology 108, 2142–2148. Brown, K. I., and Nestor, K. E. (1973). Some physiological responses of turkeys selected for high and low adrenal responses to cold stress. Poultry Sci. 52, 1948–1954. Brush, F. R., Isaacson, M. D., Pellegrino, L. J., Rykaszewski, I. M., and Shain, C. N. (1991). Characteristics of the pituitary–adrenal system in the Syracuse high- and low-avoidance strains of rats (Rattus norvegicus). Behav. Genet. 21 (1), 35–48.
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Cumming, D. C., Quigley, M. E., and Yen, S. S. C. (1983). Acute suppression of circulating testosterone levels by cortisol in men. J. Clin. Endocrinol. Metab. 57, 671–673. DeNardo, D. F., and Licht, P. (1993). Effects of corticosterone on social behavior in male lizards. Horm. Behav. 27, 184–199. Fox, S. F., Heger, N. A., and Delay, L. S. (1990). Social cost of tail loss in Uta stansburiana: Lizard tails as status-signalling badges. An. Behav. 39, 549–554. Gentsch, C., Lichtsteiner, M., Driscoll, P., and Feer, H. (1982). Differential hormonal and physiological responses to stress in Roman high- and low-avoidance rats. Physiol. Behav. 28, 259–263. Gentsch, C., Lichtsteiner, M., and Feer, H. (1988). Genetic and environmental influences on behavioral and neurochemical aspects of emotionality in rats. Experientia 44, 482–490. Hadley, M. E. (1992). ‘‘Endocrinology,’’ 3rd ed. Prentice Hall, Englewood Cliffs, NJ. Hover, E. L. (1982). ‘‘Behavioral Correlates of a Throat Color Polymorphism in a Lizard, Urosaurus ornatus.’’ University of Michigan, Ann Arbor, MI. [Ph.D. dissertation] Hover, E. L. (1985). Differences in aggressive behavior between throat color morphs in a lizard, Urosaurus ornatus. Copeia 1985, 933–940. Knapp, R. (1996). ‘‘Endocrine, Physiological and Environmental Influences on Alternative Reproductive Tactics in a Polymorphic Lizard. Arizona State University, Tempe, AZ. [Ph.D. dissertation] Knapp, R., and Moore, M. C. (1995). Hormonal responses to aggression vary in different types of agonistic encounters in male tree lizards, Urosaurus ornatus. Horm. Behav. 29, 85–105. Knapp, R., and Moore, M. C. (1996). Male morphs in tree lizards, Urosaurus ornatus, have different delayed hormonal responses to aggressive encounters. An. Behav. 52, 1045–1055. Knapp, R., and Moore, M. C. (1997). A non-invasive method for sustained elevation of steroid hormone levels in reptiles. Herp. Rev. 28, 33–36. Leshner, A. I. (1980). The interactions of experience and neuroendocrine factors in determining behavioral adaptations to aggression. Prog. Brain Res. 53, 427–438. Meaney, M. J., Viau, V., Bhatnagar, S., Betito, K., Iny, L. J., O’Donnell, D., and Mitchell, J. B. (1991). Cellular mechanisms underlying the development and expression of individual differences in the hypothalamic–pituitary–adrenal stress response. J. Steroid Biochem. Mol. Biol. 39, 265–274. Moore, M. C. (1986). Elevated testosterone levels during nonbreedingseason territoriality in a fall-breeding lizard, Sceloporus jarrovi. J. Comp. Physiol. 158, 159–163.
279 Moore, M. C., Hews, D. K., and Knapp, R. Hormonal control and evolution of alternative male phenotypes: generalizations of models for sexual differentiation. Am. Zool. [In review] Moore, M. C., and Thompson, C. W. (1990). Field endocrinology in reptiles: Hormonal control of alternative male reproductive tactics. In ‘‘Progress in Comparative Endocrinology’’ (A. Epple, C. G. Scanes, and M. H. Stetson, Eds.), pp. 685– 690. Wiley/A. R. Liss, New York. Moore, M. C., Thompson, C. W., and Marler, C. A. (1991). Reciprocal changes in corticosterone and testosterone levels following acute and chronic handling stress in the tree lizard, Urosaurus ornatus. Gen. Comp. Endocrinol. 81, 217–226. Orr, T. E., and Mann, D. R. (1992). Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis in adult male rats. Horm. Behav. 26, 350–363. Rivier, C., and Rivest, C. (1991). Effect of stress on the activity of the hypothalamic–pituitary–gonadal axis: Peripheral and central mechanisms. Biol. Reprod. 45, 523–532. Salvador, A., Martı´n, J., and Lo´pez, P. (1995). Tail loss reduces home range size and access to females in male lizards, Psammodromus algirus. Behav. Ecol. 6, 382–387. Sapolsky, R. M. (1985). Stress-induced suppression of testicular function in the wild baboon: Role of glucocorticoids. Endocrinology 116, 2273–2278. Satterlee, D. G., and Johnson, W. A. (1988). Selection of Japanese quail for contrasting blood corticosterone response to immobilization. Poultry Sci. 67, 25–32. Shire, J. G. M. (Ed.) (1979). ‘‘Genetic Variation in Hormone Systems,’’ Vol. 1. CRC Press, Boca Raton, FL. Thompson, C. W., and Moore, M. C. (1991a). Syntopic occurrence of multiple dewlap color morphs in male tree lizards, Urosaurus ornatus. Copeia 1991, 493–503. Thompson, C. W., and Moore, M. C. (1991b). Throat color reliably signals status in male tree lizards, Urosaurus ornatus. An. Behav. 42, 745–753. Thompson, C. W., and Moore, M. C. (1992). Behavioral and hormonal correlates of alternative reproductive strategies in a polygynous lizard: Tests of the relative plasticity and challenge hypotheses. Horm. Behav. 26, 568–585. Tinkle, D. W., and Dunham, A. E. (1983). Demography of the tree lizard, Urosaurus ornatus, in central Arizona. Copeia 1983, 585–598. Wingfield, J. C., O’Reilly, K. M., and Astheimer, L. B. (1995). Modulation of the adrenocortical responses to acute stress in arctic birds: A possible ecological basis. Am. Zool. 35, 285–294.
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Male Morphs in Tree Lizards Have Different Testosterone Responses to Elevated Levels of Corticosterone Rosemary Knapp1 and Michael C. Moore Department of Zoology, Arizona State University, Tempe, Arizona 85287-1501 Accepted April 16, 1997
Changes in circulating glucocorticoid and androgen levels mediate agonistic behaviors in many vertebrates. Individual variation in the magnitude of the glucocorticoid response to stressful stimuli, the negative effects of elevated glucocorticoid levels on androgen levels, or both could mediate individual differences in subsequent agonistic behavior. In a series of previous studies, we found that both alternative male reproductive morphs in the tree lizard, Urosaurus ornatus, can exhibit elevated levels of plasma corticosterone following male–male encounters, but that the territorial morph appears less likely to exhibit coincident decreases in plasma testosterone. Two studies tested the hypothesis that the two morphs differ in the degree to which testosterone levels are influenced by elevated corticosterone levels. In the first study, physically restraining males elicited endogenous elevations of circulating corticosterone levels. Testosterone levels were significantly negatively correlated with corticosterone levels in the nonterritorial morph, but there was no correlation between levels of the two steroids in territorial males. In the second study, corticosterone levels were artificially elevated in freeliving male tree lizards using a noninvasive dermal patch. This exogenous elevation of corticosterone significantly depressed testosterone levels in both morphs, but it produced a significantly greater depression in the nonterritorial morph. Nonterritorial males appear to be more sensitive than territorial males to the testosteronesuppressing effects of elevated circulating levels of 1
Present address: Section of Neurobiology and Behavior, Cornell University, Mudd Hall, Ithaca, NY 14853. 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
corticosterone. This difference between the morphs in the effects of a stress hormone on the reproductive axis may be a fundamental part of the mechanism (1) underlying behavioral tactic switching within the nonterritorial morph or (2) contributing to behavioral differences between the morphs. r 1997 Academic Press In many vertebrates, there is a negative effect of glucocorticoids on androgen production. This provides one route by which stress can potentially suppress the reproductive axis. However, the magnitude of the effect of glucocorticoids on androgens often varies considerably among individuals. Variation in these hormone responses may arise from genetic differences (Shire, 1979; Brown and Nestor, 1973; Gentsch et al., 1982, 1988; Satterlee and Johnson, 1988; Brush et al., 1991) or from different social experiences (Sapolsky, 1985; Knapp and Moore, 1996). Since both glucocorticoids and androgens are known to influence agonistic behavior (Leshner, 1980; DeNardo and Licht, 1993), individual variation in the magnitude of the negative effects of glucocorticoids on androgen levels could contribute to individual variation in agonistic behavior. Our recent work suggests the possibility that such a mechanism underlies behavioral variation between the two alternative male reproductive morphs of the tree lizard, Urosaurus ornatus. The two morphs differ in their likelihood to escalate agonistic encounters and in their territorial behavior (Hover, 1982, 1985; Thompson and Moore, 1991b, 1992). This behavioral variation
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is correlated with color variation in the dewlap (throat fan), a structure used in social communication. Males with orange dewlaps containing a central bluish-green spot (‘‘orange–blue males’’) are territorial and very likely to escalate male–male interactions. Males with orange dewlaps but lacking a central bluish-green spot (‘‘orange males’’) are nonterritorial and less likely to escalate male–male interactions. In a series of previous studies, we found that both morphs can exhibit elevated plasma levels of corticosterone 1 day following male–male encounters, but that the territorial morph appears less likely to exhibit coincident decreases in plasma testosterone (Knapp and Moore, 1995, 1996). These results suggested the hypothesis that the morphs may differ in the degree to which the hypothalamic–pituitary–gonadal (HPG) axis is suppressed by activation of the hypothalamic– pituitary–adrenal (HPA) axis. We therefore tested this hypothesis in two ways. First, we examined testosterone levels following endogenous elevation of corticosterone levels in response to restraint stress. Second, we examined the testosterone response of the two morphs to exogenous elevation of corticosterone levels. The hypothesis predicts that testosterone levels would be influenced by elevations in corticosterone to a greater extent in orange males compared with orange– blue males. The results of the two studies reported here support this prediction and, in addition, suggest that this morph difference is expressed only when corticosterone levels are elevated.
ENDOGENOUS CORTICOSTERONE ELEVATION Methods Field site. The study was conducted in Tonto National Forest within an approximately 5-km radius of the intersection of Sugarloaf Mountain–Sycamore Creek Road with Arizona State Highway 87. This area has been used regularly by our laboratory since 1986 (see Thompson and Moore, 1991a, for a full description of the site). This study was conducted between June 2 and August 6, 1988, during the active breeding season (April–August). Females may produce one to three clutches of eggs during a breeding season, and recep-
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Knapp and Moore
tive females are present throughout all but the very end of the breeding season (Tinkle and Dunham, 1983). Animal handling. Glucocorticoid levels increase in response to physical restraint in many vertebrates (e.g., Moore et al., 1991; Wingfield et al., 1995). To induce endogenous increases in corticosterone levels, male tree lizards (n 5 16 for each morph) were captured by noosing and then transferred to cloth bags for 10 or 30 min (see Moore et al., 1991). Two sampling time points were used to produce a range of corticosterone levels. A blood sample was then collected from the orbital sinus using heparinized microcapillary tubes and held on ice until return to the laboratory (within 4 hr). There the samples were centrifuged for approximately 5 min. Plasma was collected and stored at 220° until assayed. Hormone assays. Steroid hormone levels were determined by radioimmunoassay following chromatographic separation (Moore, 1986; Knapp and Moore, 1995). Prior to chromatographic separation, plasma samples were extracted in diethyl ether and then resuspended in 10% ethyl acetate in isooctane. Testosterone and corticosterone were then separated from each other and from interfering neutral lipids using celite minicolumns [a 3 g celite:1 ml ethylene glycol: propanediol mixture (1:1) over 3 g celite:1 ml water ‘‘glycol trap’’]. Each steroid was collected as a separate fraction by stepwise elution using increasing concentrations of ethyl acetate in isooctane. Fractions were dried down under nitrogen and then resuspended in buffer. Aliquots of each resuspended fraction were assayed in duplicate for each sample using radioimmunoassay. Values were corrected for individual sample recovery calculated from recovery of small amounts of radiolabeled testosterone and corticosterone that were added to each sample prior to ether extraction. Plasma samples were assayed in two assays. Intra-assay variation was 7.0% for both testosterone and corticosterone; interassay variation was 7.5% for testosterone and 11.8% for corticosterone.
Results Data were analyzed by regressing testosterone levels on corticosterone levels. Such an analysis factors out potential effects of time of the breeding season on hormone levels (Moore and Thompson, 1990). The relationship between corticosterone and testosterone differed significantly between male morphs (Fig. 1).
Testosterone Responses in Lizards
FIG. 1. Morph differences in the relationship between plasma testosterone and corticosterone levels in response to restraint stress. Lines illustrate least squares regressions and their 95% confidence intervals.
The slopes of the regressions for the two morphs differed significantly (t 5 2.73, df 5 30, P , 0.02). The slope of the log–log regression of testosterone on corticosterone was negative (20.63, r 2 5 0.70) and significantly different from zero for orange males (t 5 25.68, df 5 15, P , 0.01), but for orange–blue males was not significantly different from zero (slope 5 0.01, r 2 5 0.00; t 5 0.05, df 5 15, P 5 0.96). However, mean levels of each hormone did not differ significantly between the morphs [corticosterone (mean 6 SE): orange, 10.9 6 2.6 ng/ml; orange–blue, 16.6 6 3.8; t 5 1.25, df 5 30, P 5 0.22; testosterone: orange, 23.7 6 2.8 ng/ml; orange–blue, 23.1 6 3.6; t 5 0.12, df 5 30, P 5 0.91].
EXOGENOUS CORTICOSTERONE ELEVATION Methods The data presented in this section are part of a larger study examining multiple effects of corticosterone on male morph differences in physiology and behavior. Here we describe the portion of this larger study that was designed to test the hypothesis that the two male morphs differ in the degree of testosterone suppression following exogenous elevation of corticosterone. Animal handling. This study had two stages. First, males were captured, given an experimental manipula-
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tion, and released at the site of capture (Day 1). On the following day (Day 2), males were recaptured and a blood sample was collected. This study was conducted between May 18 and July 11, 1995, at the same field site as the study described above. Males of both morphs were captured by noosing as they were encountered on the study site on Day 1. Only males with tails that were at least 75% of their full length were included in the study because tail loss affects social interaction and home range size in other lizard species (Fox et al., 1990; Salvador et al., 1995). Following capture, males were scored for dewlap color, toe clipped for permanent identification, measured for snout–vent length (nearest mm), and weighed to the nearest 0.1 g on a portable electronic balance. Each male was also given a unique paint mark on the base of the tail to facilitate resighting on Day 2. Males were then given a dermal patch containing 4.5 µl of either (a) a solution of 3 µg corticosterone/µl sesame oil (total, 13.5 µg corticosterone; ‘‘corticosterone patch’’) or (b) sesame oil only (‘‘oil patch’’) (n 5 5–11). These patches were constructed from small pieces of adhesive bandage and vinyl electrical tape that were secured to the males’ backs by means of a transparent dressing (Johnson & Johnson Bioclusive; see Knapp and Moore, 1997, for a detailed description of the patch). Males were assigned alternately to one of the two patch treatment groups in the order they were captured. Total handling time was always less than 12 min, with almost all animals being handled for only about 5 min. Males were released at the site of capture and the location was marked with flagging and noted on a sketch map of the area. The following day (Day 2), males were resighted by searching the general vicinity of the marking site for up to 20 min. Once males were resighted, they were exposed either to an orange–blue intruder male tethered to a pole or only to the pole for a period up to 5 min. [This manipulation of exposing experimental males to an intruder male or a pole was relevant for other aspects of the larger study and is reported here only to accurately describe animal handling. Corticosterone and testosterone levels have been shown not to change within 60 min of male–male interaction in this species (Thompson and Moore, 1992; Knapp and Moore, 1995). Male–male interaction also had no significant effect on hormone levels in the present data set (t
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tests with Bonferroni correction for multiple comparisons, data not shown).] Following the behavioral test, males were again captured by noosing. Blood samples were collected from the orbital sinus using heparinized microcapillary tubes (completed within 1 min of capture) and held on ice until return to the laboratory (within 4 hr). There they were centrifuged for approximately 5 min. Plasma was collected and stored at 220° until assayed. Blood samples were also collected from an additional group of unmarked males of both morphs (‘‘no patch,’’ n 5 16–18). These males had not been handled the day before but were otherwise treated exactly as were males with patches and served as controls for possible effects of handling on hormone levels. These males were sampled on the same days that blood samples were collected from males with patches to control for abiotic factors such as weather that could potentially influence hormone levels. Hormone assays. Plasma samples were assayed for corticosterone and testosterone as described above. For this study, all samples were measured in a single assay in which intra-assay variation was 9.7% for testosterone and 5.9% for corticosterone. Statistical analyses. Hormone data were logarithmically transformed to meet assumptions of normality required for analysis of variance. Testosterone and corticosterone levels in male tree lizards are known to vary across the breeding season (Moore and Thompson, 1990). Therefore, preliminary ANCOVAs were performed with numerical day of the year (‘‘season’’) as a covariate to determine whether this was also the case in the present data set. Season was a statistically significant covariate for testosterone (F(1, 59) 5 4.56, P 5 0.037), but not corticosterone (F(1, 59) 5 0.13, P 5 0.721). Corticosterone data are presented as backtransformed means and standard errors from the analysis of variance (Fig. 2A). Testosterone data are presented as back-transformed adjusted means and standard errors from the ANCOVA to show the influence of treatment after the effect of season has been removed (Fig. 2B). Preliminary analyses also indicated that hormone levels of the two control groups did not differ for either morph (ANOVA and ANCOVA, both P . 0.13). The two control groups for each morph were therefore combined for further analyses. We present the data for the individual control groups (no patch
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Knapp and Moore
FIG. 2. Plasma corticosterone (A) and testosterone levels (B) of free-living males from the exogenous corticosterone experiment. Data are presented as back-transformed means (mean 6 SE) from analyses of variance. Hormone levels for the two control groups (no patch and oil patch; left side of figure) did not differ significantly for either morph and were therefore combined (controls) for further analysis (see text). Means with different letters for each hormone (right side of figure) are significantly different from each other, P , 0.05. Sample sizes are given between the graphs. k, orange males; b, orange–blue males.
and oil patch) in Fig. 2 for the reader’s information. Post hoc Student Newman–Kuels (SNK) tests were performed following ANOVAs to determine significant pairwise differences.
Results As expected, patch treatment had a significant effect on plasma corticosterone levels in both morphs (Table 1; Fig. 2A). Corticosterone levels of orange and orange– blue males receiving corticosterone patches were significantly elevated compared with controls (SNK tests: both P , 0.001). Corticosterone levels of the orange and orange–blue controls did not differ significantly from each other (SNK tests: P . 0.20). Corticosterone levels of orange and orange–blue males receiving corticosterone patches also did not differ (SNK test: P . 0.50). The magnitude of corticosterone elevation produced by the corticosterone patches was in the range of levels that tree lizards are capable of produc-
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Testosterone Responses in Lizards
ing endogenously under stress (Fig. 1 and Moore et al., 1991). Corticosterone patch treatment also significantly affected plasma testosterone levels (Table 1; Fig. 2B). Males of both morphs receiving corticosterone patches had significantly lower testosterone levels than controls (SNK tests: both P , 0.001). However, the morphs differed in the degree of testosterone suppression resulting from corticosterone patches; orange males had significantly lower testosterone levels than did orange–blue males (SNK test: P , 0.025). Testosterone levels of orange and orange–blue controls did not differ significantly (SNK test: P . 0.50). At the low corticosterone levels exhibited by the two control groups (no patch and oil patch), there was no significant relationship between circulating levels of corticosterone and testosterone for either morph (Fig. 3); the slopes of the log–log regressions of testosterone on corticosterone were not significantly different from zero (orange: 20.12, r 2 5 0.013; orange–blue: 5 0.11, r 2 5 0.017; both P $ 0.50).
DISCUSSION The two behavioral morphs of male tree lizards differ in the degree to which circulating testosterone levels are affected by elevation of plasma corticosterone. Testosterone levels in the less aggressive, nonter-
TABLE 1 Analysis of Variance Results for Plasma Corticosterone and Testosterone Levels for Exogenous Corticosterone Study Source Corticosterone Treatment Morph Treatment 3 morph Error Testosterone Season (covariate) Treatment Morph Treatment 3 morph Error
df
MSS
F
P
1 1 1 62
12.85 0.06 0.00 0.08
156.77 0.78 0.00
0.000 0.382 0.962
1 1 1 1 61
0.32 3.34 0.48 0.25 0.08
3.92 40.50 5.82 3.03
0.052 0.000 0.019 0.087
Note. Data were logarithmically transformed prior to analysis to meet the assumptions of analysis of variance. Season refers to the numerical day of the year.
FIG. 3. Plasma testosterone levels exhibit no significant relationship to corticosterone levels in control males (no patch and oil patch of Fig. 2) from the exogenous corticosterone study. Lines illustrate least-squares regressions and their 95% confidence intervals.
ritorial orange males were suppressed to a greater degree than were testosterone levels in the more aggressive, territorial orange–blue males. This morph difference in plasma testosterone levels occurred despite corticosterone levels being elevated equivalently in the two morphs by the experimental manipulation in each study. Further characterization of morph differences in sensitivity of testosterone levels to elevations in corticosterone requires a dose–response study. Such a component could not be included in the exogenous corticosterone study due to logistic constraints, but will be important for fully characterizing the morph difference described here. Glucocorticoids can modulate HPG axis activity at the level of the hypothalamus, pituitary, or testes in other vertebrates (Rivier and Rivest, 1991). Direct inhibitory effects of glucocorticoids at the level of the testes have been described in several species (Bambino and Hsueh, 1981; Cumming et al., 1983; Sapolsky, 1985; Orr and Mann, 1992). The site(s) of action of the suppressive effects of elevated corticosterone levels on male tree lizards, and whether this morph difference is genetic, remains to be determined. The morph difference in response of testosterone levels to corticosterone appears to exist only when corticosterone levels are elevated (compare Figs. 1 and 3). Previous studies of male tree lizards have also failed to find differences in the testosterone–corticosterone relationship for the two morphs when hormone levels are basal (Moore and Thompson, 1990; Thompson and Moore, 1992; Knapp and Moore, 1996). These
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results are reminiscent of findings that strain and experience differences in glucocorticoid levels in laboratory animals are evident only in response to moderate stressors and not under basal conditions (Gentsch et al., 1988; Meaney et al., 1991). Such differences among groups in laboratory animals may be mediated by different densities of glucocorticoid (Type II) corticosteroid receptors in certain brain regions (Meaney et al., 1991). At basal circulating levels of glucocorticoids, mineralocorticoid (Type I) receptors have a high rate of occupancy and are thus relatively insensitive to changes in corticosterone levels. Glucocorticoid (Type II) receptors, which have low occupancy rates at basal corticosteroid levels but high occupancy rates at elevated corticosteroid levels, are more responsive to elevations in circulating corticosterone. In addition, effects of corticosteroids on targets outside the HPA axis appear to be primarily mediated by glucocorticoid (Type II) receptors whereas targets within the HPA axis appear to be primarily mediated by association of corticosteroids with mineralocorticoid (Type I) receptors (Akana et al., 1992). Whether lizards possess two types of corticosteroid receptors as do mammals remains to be determined, although it seems likely given that they do secrete the mineralocorticoid aldosterone (Hadley, 1992). If tree lizards are found to possess two types of corticosteroid receptors, our data lead us to predict that orange males would possess more Type II receptors than orange–blue males in brain areas mediating HPA–HPG interactions. The greater suppression of testosterone levels in nonterritorial orange males in response to elevations in corticosterone is consistent with a hypothesized hormonal mechanism contributing to switching of reproductive tactics exhibited by these nonterritorial males. We have recently documented year-to-year variation in behavioral tactics of orange males (Knapp, 1996). When environmental conditions are relatively benign and social interactions are not particularly intense, orange males appear to adopt a sedentary satellite tactic. Under more stressful conditions that could induce elevations in circulating corticosterone, orange males apparently act as predominantly non-sitefaithful rovers. In contrast, orange–blue males exhibit the same reproductive tactic (territoriality) under all conditions. Based on the documented behavioral roles of corticosterone and testosterone, we hypothesize that
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Knapp and Moore
the elevation of corticosterone levels for orange males and the coincident decrease in testosterone levels in response to prevailing social and/or environmental stressors are part of the neuroendocrine mechanism influencing which reproductive tactic is exhibited. For orange–blue males, resistance to elevating corticosterone levels following male–male interactions (a social stressor) (Knapp and Moore, 1996), reduced testosterone suppression following elevations in corticosterone levels in response to physical or environmental stressors (this study; Knapp, 1996), or both may help to maintain aggressiveness at the level required for successful territorial maintenance (see also Moore et al., in review). Taken together our data support the speculation that a difference in the HPA axis may be an important component of the mechanism mediating the different behavioral responses of the two morphs to their social and physical environment.
ACKNOWLEDGMENTS We thank Alan Pearlman for collecting the blood samples for the endogenous corticosterone study and Diana K. Hews, Eva L. Lacy, Danika L. Painter, and Sarah K. Woodley for assistance in several aspects of the exogenous corticosterone study. This work was conducted under guidelines established by the ASU Animal Care and Use Committee and appropriate permits from Arizona Game and Fish and the United States Forest Service. Financial assistance for this work was provided by an NIMH predoctoral National Research Service Award to R.K. and NIMH MH48564 to M.C.M.
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279 Moore, M. C., Hews, D. K., and Knapp, R. Hormonal control and evolution of alternative male phenotypes: generalizations of models for sexual differentiation. Am. Zool. [In review] Moore, M. C., and Thompson, C. W. (1990). Field endocrinology in reptiles: Hormonal control of alternative male reproductive tactics. In ‘‘Progress in Comparative Endocrinology’’ (A. Epple, C. G. Scanes, and M. H. Stetson, Eds.), pp. 685– 690. Wiley/A. R. Liss, New York. Moore, M. C., Thompson, C. W., and Marler, C. A. (1991). Reciprocal changes in corticosterone and testosterone levels following acute and chronic handling stress in the tree lizard, Urosaurus ornatus. Gen. Comp. Endocrinol. 81, 217–226. Orr, T. E., and Mann, D. R. (1992). Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis in adult male rats. Horm. Behav. 26, 350–363. Rivier, C., and Rivest, C. (1991). Effect of stress on the activity of the hypothalamic–pituitary–gonadal axis: Peripheral and central mechanisms. Biol. Reprod. 45, 523–532. Salvador, A., Martı´n, J., and Lo´pez, P. (1995). Tail loss reduces home range size and access to females in male lizards, Psammodromus algirus. Behav. Ecol. 6, 382–387. Sapolsky, R. M. (1985). Stress-induced suppression of testicular function in the wild baboon: Role of glucocorticoids. Endocrinology 116, 2273–2278. Satterlee, D. G., and Johnson, W. A. (1988). Selection of Japanese quail for contrasting blood corticosterone response to immobilization. Poultry Sci. 67, 25–32. Shire, J. G. M. (Ed.) (1979). ‘‘Genetic Variation in Hormone Systems,’’ Vol. 1. CRC Press, Boca Raton, FL. Thompson, C. W., and Moore, M. C. (1991a). Syntopic occurrence of multiple dewlap color morphs in male tree lizards, Urosaurus ornatus. Copeia 1991, 493–503. Thompson, C. W., and Moore, M. C. (1991b). Throat color reliably signals status in male tree lizards, Urosaurus ornatus. An. Behav. 42, 745–753. Thompson, C. W., and Moore, M. C. (1992). Behavioral and hormonal correlates of alternative reproductive strategies in a polygynous lizard: Tests of the relative plasticity and challenge hypotheses. Horm. Behav. 26, 568–585. Tinkle, D. W., and Dunham, A. E. (1983). Demography of the tree lizard, Urosaurus ornatus, in central Arizona. Copeia 1983, 585–598. Wingfield, J. C., O’Reilly, K. M., and Astheimer, L. B. (1995). Modulation of the adrenocortical responses to acute stress in arctic birds: A possible ecological basis. Am. Zool. 35, 285–294.
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