Effects of testosterone on urogenital tract morphology and androgen receptor expression in immature Eastern Fence lizards (Sceloporus undulatus).

In non-avian reptiles, the onset of sexual dimorphism of the major structures of the urogenital tract varies temporally relative to gonadal differentiation, more so than in other amniote lineages. In the current study, we used tonic-release implants to investigate the effects of exogenous testosterone (T) on postnatal development of the urogenital tract in juvenile Eastern Fence Lizards (Sceloporus undulatus) to better understand the mechanisms underlying the ontogeny of sexual differentiation in reptiles. We examined gonads, mesonephric kidneys and ducts (male reproductive tract primordia), paramesonephric ducts (oviduct primordia), sexual segments of the kidneys (SSKs), and hemiphalluses to determine which structures were sexually dimorphic independent of T treatment and which structures exhibited sexually dimorphic responses to T. To better understand tissue-level responsiveness to T treatment, we also characterized androgen receptor (AR) expression by immunohistochemistry. At approximately 4 months after hatching in control animals, gonads were well differentiated but quiescent; paramesonephric ducts had fully degenerated in males; mesonephric kidneys, mesonephric ducts, and SSKs remained sexually undifferentiated; and hemiphalluses could not be everted in either sex. Exogenous T caused enlargement, regionalization, and secretory activity of the mesonephric ducts and SSKs in both sexes; enlargement and regionalization of the oviducts in females; and enlargement of male hemipenes. The most responsive tissues exhibited moderate but diffuse staining for AR in control lizards and intense nuclear staining in T-treated lizards, suggestive of autoregulation of AR. The similarity between sexes in the responsiveness of the mesonephric ducts and SSK to T indicates an absence of sexually dimorphic organizational effects in these structures prior to treatment, which was initiated approximately 2 months after hatching. In contrast, the sex-specific responses in oviducts and hemipenes indicate that significant organization and/or differentiation had taken place prior to treatment.

Chigger mite (Eutrombicula alfreddugesi) ectoparasitism does not contribute to sex differences in growth rate in eastern fence lizards (Sceloporus undulatus).

Parasitism is nearly ubiquitous in animals and is frequently associated with fitness costs in host organisms, including reduced growth, foraging, and reproduction. In many species, males tend to be more heavily parasitized than females and thus may bear greater costs of parasitism. Sceloporus undulatus is a female-larger, sexually size dimorphic lizard species that is heavily parasitized by chigger mites (Eutrombicula alfreddugesi). In particular, the intensity of mite parasitism is higher in male than in female juveniles during the period of time when sex differences in growth rate lead to the development of sexual size dimorphism (SSD). Sex-biased differences in fitness costs of parasitism have been documented in other species. We investigated whether there are growth costs of mite ectoparasitism, at a time coinciding with sex differences in growth rate and the onset of SSD. If there are sex-biased growth costs of parasitism, then this could suggest a contribution to the development of SSD in S. undulatus. We measured growth and mite loads in two cohorts of unmanipulated, field-active yearlings by conducting descriptive mark-recapture studies during the activity seasons of 2016 and 2019. Yearling males had consistently higher mid-summer mite loads and consistently lower growth rates than females. However, we found that growth rate and body condition were independent of mite load in both sexes. Furthermore, growth rates and mite loads were higher in 2019 than in 2016. Our findings suggest that juveniles of S. undulatus are highly tolerant of chigger mites and that any costs imposed by mites may be at the expense of functions other than growth. We conclude that sex-biased mite ectoparasitism does not contribute to sex differences in growth rate and, therefore, does not contribute to the development of SSD.

Species differences in hormonally mediated gene expression underlie the evolutionary loss of sexually dimorphic coloration in Sceloporus lizards.

Phenotypic sexual dimorphism often involves the hormonal regulation of sex-biased expression for underlying genes. However, it is generally unknown whether the evolution of hormonally mediated sexual dimorphism occurs through upstream changes in tissue sensitivity to hormone signals, downstream changes in responsiveness of target genes, or both. Here, we use comparative transcriptomics to explore these possibilities in 2 species of Sceloporus lizards exhibiting different patterns of sexual dichromatism. Sexually dimorphic S. undulatus develops blue and black ventral coloration in response to testosterone, while sexually monomorphic S. virgatus does not, despite exhibiting similar sex differences in circulating testosterone levels. We administered testosterone implants to juveniles of each species and used RNAseq to quantify gene expression in ventral skin. Transcriptome-wide responses to testosterone were stronger in S. undulatus than in S. virgatus, suggesting species differences in tissue sensitivity to this hormone signal. Species differences in the expression of genes for androgen metabolism and sex hormone-binding globulin were consistent with this idea, but expression of the androgen receptor gene was higher in S. virgatus, complicating this interpretation. Downstream of androgen signaling, we found clear species differences in hormonal responsiveness of genes related to melanin synthesis, which were upregulated by testosterone in S. undulatus, but not in S. virgatus. Collectively, our results indicate that hormonal regulation of melanin synthesis pathways contributes to the development of sexual dimorphism in S. undulatus, and that changes in the hormonal responsiveness of these genes in S. virgatus contribute to the evolutionary loss of ventral coloration.

Invited review: Adrenocortical function in avian and non-avian reptiles: Insights from dispersed adrenocortical cells.

Herein we review our work involving dispersed adrenocortical cells from several lizard species: the Eastern Fence Lizard (Sceloporus undulatus), Yarrow's Spiny Lizard (Sceloporus jarrovii), Striped Plateau Lizard (Sceloporus virgatus) and the Yucatán Banded Gecko (Coleonyx elegans). Early work demonstrated changes in steroidogenic function of adrenocortical cells derived from adult S. undulatus associated with seasonal interactions with sex. However, new information suggests that both sexes operate within the same steroidogenic budget over season. The observed sex effect was further explored in orchiectomized and ovariectomized lizards, some supported with exogenous testosterone. Overall, a suppressive effect of testosterone was evident, especially in cells from C. elegans. Life stage added to this complex picture of adrenal steroidogenic function. This was evident when sexually mature and immature Sceloporus lizards were subjected to a nutritional stressor, cricket restriction/deprivation. There were divergent patterns of corticosterone, aldosterone, and progesterone responses and associated sensitivities of each to corticotropin (ACTH). Finally, we provide strong evidence that there are multiple, labile subpopulations of adrenocortical cells. We conclude that the rapid (days) remodeling of adrenocortical steroidogenic function through fluctuating cell subpopulations drives the circulating corticosteroid profile of Sceloporus lizard species. Interestingly, progesterone and aldosterone may be more important with corticosterone serving as essential supportive background. In the wild, the flux in adrenocortical cell subpopulations may be adversely susceptible to climate-change related disruptions in food sources and to xenobiotic/endocrine-disrupting chemicals. We urge further studies using native lizard species as bioindicators of local pollutants and as models to examine the broader eco-exposome.

Tick abundance and diversity are substantially lower in thinned vs. unthinned forests in the New Jersey Pinelands National Reserve, USA.

Forest thinning is a management tool used in the New Jersey Pinelands and elsewhere to improve forest health and resilience, mitigate wildfire risk, and manage for wildlife. Forest thinning leads to warmer drier microclimates, which have been shown in both field and laboratory studies to reduce tick survival and reproduction. To directly assess the effects of forest thinning on the abundance and diversity of ticks and on the prevalence of tick-borne human pathogens, we sampled ticks weekly from March to November 2021 at three replicated pairs of thinned and unthinned forest sites composed primarily of pitch-pine, shortleaf pine, and various oak species. We characterized microclimate in the understory and forest floor at each sampling plot by deploying multiple data loggers to monitor temperature and relative humidity throughout the study period. As expected, we found that thinned plots were significantly drier and warmer than unthinned plots. We also found that average questing tick abundance was 92% lower in thinned as compared with unthinned plots. Of the three main tick species collected in unthinned plots (Amblyomma americanum, Ixodes scapularis, and Dermacentor albipictus) only A. americanum and a single I. scapularis were collected in thinned plots. Prevalence of Ehrlichia species in A. americanum did not differ between treatments, and the sole I. scapularis collected in a thinned plot was infected with Borrelia burgdorferi sensu lato. However, the significant and much lower tick abundance in thinned plots indicates a lower risk of human-tick encounters. Our results add to the growing evidence that landscape and forest management can reduce local tick abundance, thereby reducing tick-borne disease risk.

A chromosome-level genome assembly for the eastern fence lizard (Sceloporus undulatus), a reptile model for physiological and evolutionary ecology.

BACKGROUND: High-quality genomic resources facilitate investigations into behavioral ecology, morphological and physiological adaptations, and the evolution of genomic architecture. Lizards in the genus Sceloporus have a long history as important ecological, evolutionary, and physiological models, making them a valuable target for the development of genomic resources. FINDINGS: We present a high-quality chromosome-level reference genome assembly, SceUnd1.0 (using 10X Genomics Chromium, HiC, and Pacific Biosciences data), and tissue/developmental stage transcriptomes for the eastern fence lizard, Sceloporus undulatus. We performed synteny analysis with other snake and lizard assemblies to identify broad patterns of chromosome evolution including the fusion of micro- and macrochromosomes. We also used this new assembly to provide improved reference-based genome assemblies for 34 additional Sceloporus species. Finally, we used RNAseq and whole-genome resequencing data to compare 3 assemblies, each representing an increased level of cost and effort: Supernova Assembly with data from 10X Genomics Chromium, HiRise Assembly that added data from HiC, and PBJelly Assembly that added data from Pacific Biosciences sequencing. We found that the Supernova Assembly contained the full genome and was a suitable reference for RNAseq and single-nucleotide polymorphism calling, but the chromosome-level scaffolds provided by the addition of HiC data allowed synteny and whole-genome association mapping analyses. The subsequent addition of PacBio data doubled the contig N50 but provided negligible gains in scaffold length. CONCLUSIONS: These new genomic resources provide valuable tools for advanced molecular analysis of an organism that has become a model in physiology and evolutionary ecology.

Functional remodeling of adrenal steroidogenic tissue by food deprivation in the lizard, Sceloporus undulatus.

The present study examined how food availability interacts with age to modulate lizard adrenal steroidogenic function at the cellular level. Adult male and juvenile male and female Eastern Fence Lizards (Sceloporus undulatus) underwent a period of food deprivation with or without a shorter re-feeding period. Lizards maintained on a full feeding regimen served as the controls. Across the feeding regimens, plasma corticosterone of adult lizards was unchanged whereas that of food-deprived juvenile lizards was increased nearly 7 times and this increase was normalized by a short re-feeding period. Freshly dispersed adrenocortical cells derived from these lizards were incubated with ACTH and the production of selected steroids was measured by highly specific radioimmunoassay. Net maximal steroid rates of juvenile cells were 161% greater than those of adult cells. Adult and juvenile progesterone rates were consistently suppressed by food deprivation (by nearly 48%) and were normalized by a re-feeding period, whereas divergent effects were seen with corticosterone and aldosterone rates. Food deprivation suppressed corticosterone rates of adult cells by 43% but not those of juvenile cells. In a reciprocal manner, food deprivation had no significant effect on aldosterone rates of adult cells, but it suppressed those of juvenile cells by 52%. A short re-feeding period normalized most rates in both adult and juvenile cells and further augmented the adult aldosterone rate by 54%. The effect of the feeding regimens on ACTH sensitivity varied with life stage and with steroid. The overall sensitivity of adult cells to ACTH was nearly three times that of juvenile cells. Collectively, the data presented here and data from previous work indicate that food restriction/deprivation in Sceloporus lizard species causes a functional remodeling of the adrenocortical tissue. Furthermore, life stage adds more complexity to this remodeling.

Living on the edge: Glucocorticoid physiology in desert iguanas (Dipsosaurus dorsalis) is predicted by distance from an anthropogenic disturbance, body condition, and population density.

Ecological factors, such as habitat quality, influence the survival and reproductive success of free-living organisms. Urbanization, including roads, alters native habitat and likely influences physiology, behavior, and ultimately Darwinian fitness. Some effects of roads are clearly negative, such as increased habitat fragmentation and mortality from vehicle collision. However, roads can also have positive effects, such as decreasing predator density and increased vegetation cover, particularly in xeric habitats due to increased water run-off. Glucocorticoids are metabolic hormones that reflect baseline metabolic needs, increase in response to acute challenges, and may mediate endogenous resource trade-offs between survival and reproduction. Here we examined circulating concentrations of corticosterone (baseline and stress-induced) in desert iguanas (Dipsosaurus dorsalis) in relation to the distance from a major anthropogenic disturbance, a high-traffic road in Palm Springs, CA. Additionally, we analyzed body condition and population density as additional predictors of glucocorticoid physiology. Surprisingly, we found lower baseline CORT levels closer to the road, but no effect of distance from road on stress-induced CORT or stress responsiveness (difference between baseline and stress-induced concentrations). Both population density and body condition were negative predictors of baseline CORT, stress-induced CORT, and stress responsiveness. Given the known effect of roads to increase run-off and vegetation density, increased water availability may improve available forage and shade, which may then increase the carrying capacity of the habitat and minimize metabolic challenges for this herbivorous lizard. However, it is important to recognize that surfaces covered by asphalt are not usable habitat for iguanas, likely resulting in a net habitat loss.

Testosterone Reduces Growth and Hepatic IGF-1 mRNA in a Female-Larger Lizard, Sceloporus undulatus: Evidence of an Evolutionary Reversal in Growth Regulation.

Previous research has demonstrated that testosterone (T) can inhibit growth in female-larger species and stimulate growth in male-larger species, but the underlying mechanisms of this regulatory bipotentiality have not been investigated. In this study, we investigated the effects of T on the expression of hepatic insulin-like growth factor-1 (IGF-1) mRNA and circulating IGF-1 hormone in Sceloporus undulatus, a species of lizard in which females grow faster to become larger than males and in which T inhibits growth. Experiments were performed in captivity on mature female and male adults in the asymptotic phase of their growth curve and on actively growing, pre-reproductive juveniles. In adult males, the expression of hepatic IGF-1 mRNA increased following surgical castration and returned to control levels with T replacement; in intact adult females, exogenous T had no effect on IGF-1 mRNA expression. In juveniles, T significantly reduced both growth and the expression of hepatic IGF-1 mRNA to similar extents in intact females and in castrated males. The relative inhibitory effects of T on mRNA expression were greater in juveniles than in adults. Plasma IGF-1 hormone was about four times higher in juveniles than in adults, but T had no significant effect on IGF-1 hormone in either sex or in either age group. Our finding of inhibition of the expression of hepatic IGF-1 mRNA stands in contrast to the stimulatory effects of T in the published body of literature. We attribute our novel finding to our use of a species in which T inhibits rather than stimulates growth. Our findings begin to explain how T has the regulatory bipotentiality to be stimulatory in some species and inhibitory in others, requiring only an evolutionary reversal in the molecular regulation of growth-regulatory genes including IGF-1. Further comparative transcriptomic studies will be required to fully resolve the molecular mechanism of growth inhibition.

Modulation of adrenal steroidogenesis by testosterone in the lizard, Coleonyx elegans.

Our previous work with adrenocortical cells from several Sceloporus lizard species suggests that gonadal hormones influence the steroidogenic capacity and the sensitivity to ACTH. However, there are discrepancies in these cellular response parameters suggesting that the effects of gonadal hormones on adrenocortical function vary with species, sex, age, season, and environmental/experimental conditions. To gain further insight into these complex interactions, here we report studies on Coleonyx elegans, Eublepharidae (Yucatán Banded Gecko), which is only distantly related to Sceloporus lizards via a basal common ancestor and in captivity, reproduces throughout the year. We hypothesized that a more constant reproductive pattern would result in less variable effects of gonadal hormones on adrenocortical function. Reproductively mature male geckos were orchiectomized with and without replacement of testosterone (300 µg) via an implanted Silastic® tube. Reproductively mature intact female geckos received implants with and without testosterone. After 11 weeks, adrenocortical cells were isolated from these lizards and incubated with corticotropin (ACTH) for 3 h at 28 °C. Three adrenocortical steroids, progesterone, corticosterone and aldosterone, were measured by highly specific radioimmunoassays. The production rate of each steroid was statistically analyzed using established software and net maximal rate (by subtracting the basal rate) in response to ACTH was determined. In general, corticosterone predominated and comprised ∼83% of the total net maximal rate, followed by progesterone (∼14%) and aldosterone (∼3%). Compared to the functional responses of adrenocortical cells derived from other lizards thus far, adrenocortical cells from C. elegans exhibited a depressed steroid response to ACTH and this depressed response was more pronounced in male cells. In addition, other sex differences in cellular response were apparent. In female cells, the net maximal rates of progesterone, corticosterone and aldosterone were, respectively, 161, 122 and 900% greater than those in intact-male cells. In contrast, cellular sensitivity to ACTH, as determined by the half-maximally effective steroidogenic concentration (EC50) of ACTH, did not differ between intact-male and intact-female adrenocortical cells. Treatment effects were most striking for corticosterone, the putative, major glucocorticoid in lizards. Orchiectomy caused an increase in the net maximal corticosterone rate equivalent to that of intact-female cells. Testosterone maintenance in orchiectomized lizards completely suppressed the stimulatory effect of orchiectomy. However, orchiectomy with or without testosterone maintenance did not alter cellular sensitivity to ACTH. The effect of testosterone supplementation in intact females, although suppressive, was notably different from its effect in orchiectomized males. Its effect on the net maximal corticosterone rate was relatively modest and did not completely "masculinize" the greater rate seen in intact-female cells. However, testosterone supplementation dramatically suppressed the basal corticosterone rate (by 82%) and enhanced the overall cellular sensitivity to ACTH by 150%, two effects not seen in cells derived from testosterone-treated orchiectomized lizards. Collectively, these findings clearly indicating that the gonad directly or indirectly regulates lizard adrenocortical cell function. Whereas other gonadal or extra-gonadal factors may play a role, testosterone appears to be an essential determinant of the observed sex differences in adrenocortical function.

Sex hormones and the development of sexual size dimorphism: 5α-dihydrotestosterone inhibits growth in a female-larger lizard (Sceloporus undulatus).

Sexual differences in adult body size [sexual size dimorphism (SSD)] and color (sexual dichromatism) are widespread, and both male- and female-biased dimorphisms are observed even among closely related species. A growing body of evidence indicates testosterone can regulate growth, thus the development of SSD, and sexual dichromatism. However, the mechanism(s) underlying these effects are conjectural, including possible conversions of testosterone to estradiol (E2) or 5α-dihydrotestosterone (DHT). In the present study, we hypothesized that the effects of testosterone are physiological responses mediated by androgen receptors, and we tested two specific predictions: (1) that DHT would mimic the effects of testosterone by inhibiting growth and enhancing coloration, and (2) that removal of endogenous testosterone via surgical castration would stimulate growth. We also hypothesized that females share downstream regulatory networks with males and predicted that females and males would respond similarly to DHT. We conducted experiments on eastern fence lizards (Sceloporus undulatus), a female-larger species with striking sexual dichromatism. We implanted Silastic® tubules containing 150 µg DHT into intact females and intact and castrated males. We measured linear growth rates and quantified color for ventral and dorsal surfaces. We found that DHT decreased growth rate and enhanced male-typical coloration in both males and females. We also found that, given adequate time, castration alone is sufficient to stimulate growth rate in males. The results presented here suggest that: (1) the effects of testosterone on growth and coloration are mediated by androgen receptors without requiring aromatization of testosterone into E2, and (2) females possess the androgen-receptor-mediated regulatory networks required for initiating male-typical inhibition of growth and enhanced coloration in response to androgens.

Androgenic control of male-typical behavior, morphology and sex recognition is independent of the mode of sex determination: A case study on Lichtenfelder's gecko (Eublepharidae: Goniurosaurus lichtenfelderi).

Previous work on lizards has shown that many sexually dimorphic traits depend on testosterone (T), but the details of this control can vary among species. Here, we tested the role of T on the expression of morphological, physiological, and behavioral traits in Lichtenfelder's gecko (Goniurosaurus lichtenfelderi), from the lizard family Eublepharidae notable for interspecific variation in sexually dimorphic traits and the mode of sex determination. Experiments included three groups of males (intact control, surgically castrated, castrated with T replacement) and two groups of females (intact control, T supplemented). In males, castration caused reductions in 1) the size of hemipenes, 2) offensive aggression, 3) male sexual behavior in a neutral arena, 4) activity of precloacal glands, and 5) loss of male chemical cues for sex recognition. These reductions were not observed in castrated males with T replacement. Interestingly, castrated males performed sexual behavior in their home cages, which shows that the effect of T depends on the environmental context. Notably, tail vibration, previously reported as a courtship behavior in other eublepharids, is displayed by males of G. lichtenfelderi during interactions with conspecifics of both sexes, suggesting an evolutionary shift in the meaning of this signal. In females, T induced growth of hemipenes and male-typical courtship but did not induce precloacal pore activity, aggression, or mounting. In comparison to previous reports on Eublepharis macularius, our results indicate that effects of T do not depend on the mode of sex determination. Further, our results extend our understanding of the complexity of control of male traits and illustrate how lability in the effects of T can be a general mechanism causing evolutionary changes in the components of suites of functionally correlated traits.

Nutritional modulation of IGF-1 in relation to growth and body condition in Sceloporus lizards.

Nutrition and energy balance are important regulators of growth and the growth hormone/insulin-like growth factor (GH/IGF) axis. However, our understanding of these functions does not extend uniformly to all classes of vertebrates and is mainly limited to controlled laboratory conditions. Lizards can be useful models to improve our understanding of the nutritional regulation of the GH/IGF-1 axis because many species are relatively easy to observe and manipulate both in the laboratory and in the field. In the present study, the effects of variation in food intake on growth, body condition, and hepatic IGF-1 mRNA levels were measured in (1) juveniles of Sceloporus jarrovii maintained on a full or 1/3 ration and (2) hatchlings of Sceloporus undulatus subjected to full or zero ration with or without re-feeding. These parameters plus plasma IGF-1 were measured in a third experiment using adults of S. undulatus subjected to full or zero ration with or without re-feeding. In all experiments, plasma corticosterone was measured as an anticipated indicator of nutritional stress. In S. jarrovii, growth and body condition were reduced but lizards remained in positive energy balance on 1/3 ration, and hepatic IGF-1 mRNA and plasma corticosterone were not affected in comparison to full ration. In S. undulatus, growth, body condition, hepatic IGF-1 mRNA, and plasma IGF-1 were all reduced by zero ration and restored by refeeding. Plasma corticosterone was increased in response to zero ration and restored by full ration in hatchlings but not adults of S. undulatus. These data indicate that lizards conform to the broader vertebrate model in which severe food deprivation and negative energy balance is required to attenuate systemic IGF-1 expression. However, when animals remain in positive energy balance, reduced food intake does not appear to affect systemic IGF-1. Consistent with other studies on lizards, the corticosterone response to reduced food intake is an unreliable indicator of nutritional stress. Further studies on ecologically relevant variation in food intake are required to establish the importance of nutrition as an environmental regulator of the GH/IGF axis. Within the range of positive energy balance, the potential involvement of molecular signals in growth regulation requires further investigation.

Elevated testosterone is required for male copulatory behavior and aggression in Madagascar ground gecko (Paroedura picta).

Elevated levels of gonadal androgens are often required for the expression of male-specific behavioral and morphological traits in all classes of vertebrates, including reptiles. Here, we tested the role of male gonadal androgens in the control of male sexual behavior, aggressive behavior, and size of the hemipenes in the gecko Paroedura picta. We performed hormonal manipulations involving castration with and without testosterone (T) replacement in males and application of exogenous T and ovariectomy in females. Castration suppressed sexual behavior and hemipenes size in males, and these effects were fully rescued by exogenous T. Sexual behavior and growth of the hemipenes were masculinized by male-typical levels of T in females, while ovariectomized females retained female-typical expression of these traits. These results indicate that the development of male sexual behavior in adult females does not require early or pubertal organization. Elevated T increased the likelihood of aggressive behavior directed toward a male intruder, but aggression occurred only rarely. Elevated T is necessary and sufficient for enlargement of the hemipenes and the expression of male sexual behavior in both males and females of Paroedura picta. In contrast to sexual behavior, the expression of aggressive behavior is apparently more dependent on other factors in addition to T itself.

Ontogeny of pronounced female-biased sexual size dimorphism in the Malaysian cat gecko (Aeluroscalabotes felinus: Squamata: Eublepharidae): a test of the role of testosterone in growth regulation.

Species differences in the effect of male gonadal androgens on male growth are considered a possible mechanism allowing shifts in magnitude and even direction of sexual size dimorphism (SSD) in squamate reptiles. According to the bipotential growth regulation hypothesis, the androgen testosterone (T) enhances male growth in species with male-biased SSD and conversely inhibits male growth in males of female-larger species. In the present study, we describe the ontogeny of the pronounced female-biased SSD and report the effect of T on growth via hormonal manipulations in males and females of the Malaysian cat gecko (Aeluroscalabotes felinus). In accord with the predictions of the bipotential growth regulation hypothesis, growth was inhibited by replacement of T in castrated males. Additionally, exogenous T inhibited growth of females to male-typical levels. Nevertheless, male castration alone did not significantly affect growth, contrary to the prediction of the bipotential growth regulation hypothesis, which contradicts the generality of this hypothesis. Application of exogenous T to females can interfere with normal ovarian function. Therefore, although not directly tested in this study, we suggest that ovarian effects on the ontogeny of SSD in A. felinus are consistent with our results. The development of SSD is a function of differential growth between the sexes, and potential sex-specific growth regulation in both males and females should be taken into account as possible proximate mechanisms responsible for SSD.

Effects of food restriction on steroidogenesis in dispersed adrenocortical cells from Yarrow's Spiny Lizard (Sceloporus jarrovii).

Changes in energy balance can lead to functional alterations at all levels of the hypothalamic-pituitary-adrenal (HPA) axis. However, relatively little is known about how energy balance affects functional properties of adrenocortical cells themselves. We investigated effects of restricted food intake on sensitivity to ACTH and rates of steroidogenesis in adrenocortical cells isolated from growing female and male Yarrow's Spiny Lizards (Sceloporus jarrovii). At the end of the feeding regimen, we assayed acute (3h) progesterone (P(4)), corticosterone (B), and aldosterone (ALDO) production in response to ACTH in dispersed adrenocortical cells. Food restriction depressed growth rate by about 50% in both males and females but did not alter baseline plasma B measured at 10 weeks in either sex. At the cellular level, food restriction had the following effects: (1) increased basal B production in both sexes and basal ALDO production in males, (2) increased net maximal rates of production of P(4), B, and ALDO in response to ACTH, and (3) no overall effect on adrenocortical cellular sensitivity to ACTH. There were modest sex differences: overall rates of P(4) production were 46% greater in cells from females than from males, and in response to food restriction, the net maximal rate of ALDO production was 50% greater in cells from males than from females. Our results demonstrate that food restriction in S. jarrovii increases adrenocortical cellular rates of steroid production without affecting overall cellular sensitivity to ACTH.

Male sexual behavior does not require elevated testosterone in a lizard (Coleonyx elegans, Eublepharidae).

Male sexual behavior depends on gonadal androgens in species of all major vertebrate lineages, including reptiles. However, male sexual behavior includes distinct appetitive and consummatory phases, typically denoted as courtship and mounting, with potentially different hormonal control. Different proximate controls of courtship versus mounting could enable disconnected evolutionary losses and gains of various aspects of male sexual behavior. Male courtship display, which is activated by testosterone (T) in many species, is an ancestral trait in the lizard family Eublepharidae. However, Coleonyx elegans (Yucatan Banded Gecko) lost the courtship display, while retaining a highly simplified male sexual behavior that involves only mounting for copulation. We performed surgical manipulations (castration with and without T replacement in adult males; implantation of adult females with exogenous T) to investigate hormonal mechanisms involved in this evolutionary novelty. Our results indicate that the expression of simplified sexual behavior in C. elegans does not require elevated circulating levels of T, a finding that is previously unreported in lizards. In females, however, exogenous T induced male-like mounting. Thus, the mounting phase of sexual behavior is not activated by T in the traditional sense of this term but probably requires post-natal, maturational organization (if not periodic reorganization) by androgens. We conclude that the simplification of male sexual behavior and its independence from elevated levels of circulating androgens in C. elegans evolved via 1) evolutionary loss of the androgen-activated courtship display and 2) retention of the mounting phase, which has a longer "functional memory" for the effects of androgenic steroids.

Hormones, performance and fitness: Natural history and endocrine experiments on a lizard (Sceloporus undulatus).

We used the "morphology-performance-fitness" paradigm (Arnold, 1983) as our framework to investigate endocrine control of performance and fitness in Sceloporus undulatus (Eastern Fence Lizard). Focusing on males, we used the "natural experiments" of seasonal, sexual, and developmental variation in growth and in exercise endurance to identify testosterone and corticosterone as potential modulators of performance and related traits of interest. We followed with experimental manipulations of testosterone to investigate functional relationships, both in the laboratory and in the field. Further, we used focal observations and demographic studies, coupled with genetic determination of paternity, to test associations between performance and fitness, measured as reproductive success. We found that in males, endurance and plasma concentrations of testosterone and corticosterone are at their peaks in the spring breeding season, when lizards are most actively engaged in patrolling home ranges and in reproductive behavior. At that time, plasma concentrations of testosterone are correlated with body size; plasma concentrations of corticosterone and parameters of home range, including area and the number of overlapped females, are correlated with home-range overlap between males and females. During prereproductive development, males (but not females) experience a maturational increase in plasma testosterone. At about the same time, they become more active, expand their home ranges, and grow less quickly than do females, suggesting a trade-off in the allocation of energy, mediated by testosterone. Experimentally, testosterone has positive effects on fitness by stimulating endurance and reproductive activity and increasing home-range area, but it exacts costs in fitness by increasing ectoparasitism, decreasing growth, and decreasing survivorship. We found evidence of selection on body size, endurance, and home-range size (and thus access to potential mates). Despite having positive effects on performance traits, plasma concentrations of testosterone were not correlated with number of offspring sired by males. However, we found a strong correlation between the level of plasma corticosterone and the number of offspring sired. We also found evidence of size-assortative mating, indicating that for males, both the number and the size (and thus, fecundity) of their mates increase with body size. Our studies exemplify the power of natural history combined with experimental endocrine manipulations to identify hormonal regulators of performance and linkages to fitness. Furthermore, our results illustrate ecological and evolutionary significance of individual variation in endocrine traits.

Gonadal modulation of in vitro steroidogenic properties of dispersed adrenocortical cells from Sceloporus lizards.

Effects of adrenal corticosteroids on reproductive and endocrine functions of the gonads are well known, but reciprocal effects of gonadal hormones on the hypothalamo-pituitary-adrenal (HPA) axis and on adrenocortical steroidogenesis in particular have received much less attention. We investigated effects of gonadectomy and testosterone (T) replacement on adrenocortical cell function in a year-long field study of male Sceloporus undulatus (Eastern Fence Lizard) and in a shorter term laboratory study with male Sceloporus jarrovii (Yarrow's Spiny Lizard). We also compared females to males in Sceloporus virgatus (Striped Plateau Lizard) and investigated effects of gonadectomy in short-term laboratory experiment on females of this species. As measured by in vitro production of progesterone (P(4)), corticosterone (B), and aldosterone (ALDO), sensitivity of adrenocortical cells to corticotrophin (ACTH) was lower in control males than females of S. virgatus. In S. jarrovii males, cellular sensitivity to ACTH was reduced by orchiectomy but was not restored to levels of intact controls by T replacement. By contrast, in S. undulatus, cellular sensitivity to ACTH was not affected by orchiectomy alone but was reduced by T replacement in orchiectomized males. Maximal rates of steroid production were less consistently affected by experimental treatments, but were lower in males than in females of S. virgatus and were dramatically reduced by T replacement in orchiectomized S. undulatus males. Overall, our experiments clearly demonstrate two distinct sources of variation in functional capacities of dispersed adrenocortical cells isolated from Sceloporus lizards: (1) naturally occurring differences between males and females (Carsia and John-Alder, 2003), and (2) species-dependent changes in response to surgical gonadectomy with or without exogenous testosterone. Sex differences and functional lability in adrenocortical cells are probably widespread among vertebrates and may be an important component of variation in output of the HPA.

Testosterone stimulates the expression of a social color signal in Yarrow's Spiny Lizard, Sceloporus jarrovii.

The sex steroid testosterone has been shown to regulate the development of male-specific coloration in many organisms that exhibit sexual dichromatism, but the role of testosterone is less certain for species in which both sexes express bright coloration. Lizards in the genus Sceloporus possess bright blue patches on their throats and abdomens. These patches, which are used in social signaling, are often regulated by testosterone and are consequently expressed only in males of most species. However, Yarrow's Spiny Lizard (Sceloporus jarrovii, Cope 1875) exhibits a derived condition in which both sexes express bright blue ventral patches despite dramatic sexual differences in circulating testosterone levels throughout postnatal ontogeny. In this study, we used surgical castration and hormone replacement in juvenile males to test the hypothesis that testosterone stimulates the expression of blue ventral coloration in S. jarrovii. In two separate experiments conducted in captivity and the natural field environment, we found that surgical castration decreased the hue and saturation while increasing the brightness of blue throat and abdominal patches. Castration also decreased the amount of black pigment bordering the blue throat patch. Treatment of castrated males with exogenous testosterone restored all aspects of ventral coloration to values similar to those of intact control males. Early organizational effects of testosterone during prenatal development may lead to the expression of blue coloration in both sexes, but the results of our present experiments indicate that subsequent effects of testosterone during sexual maturation further enhance the coloration of males.