Male Green Anoles Research Articles

Social boldness correlates with brain gene expression in male green anoles

Within populations, some individuals tend to exhibit a bold or shy social behavior phenotype relative to the mean. The neural underpinnings of these differing phenotypes – also described as syndromes, personalities, and coping styles – is an area of ongoing investigation. Although a social decision-making network has been described across vertebrate taxa, most studies examining activity within this network do so in relation to exhibited differences in behavioral expression. Our study instead focuses on constitutive gene expression in bold and shy individuals by isolating baseline gene expression profiles that influence social boldness predisposition, rather than those reflecting the results of social interaction and behavioral execution. We performed this study on male green anole lizards (Anolis carolinensis), an established model organism for behavioral research, which provides a crucial comparison group to investigations of birds and mammals. After identifying subjects as bold or shy through repeated reproductive and agonistic behavior testing, we used RNA sequencing to compare gene expression profiles between these groups within various forebrain, midbrain, and hindbrain regions. The ventromedial hypothalamus had the largest group differences in gene expression, with bold males having increased expression of neuroendocrine and neurotransmitter receptor and calcium channel genes compared to shy males. Conversely, shy males express more integrin alpha-10 in the majority of examined regions. There were no significant group differences in physiology or hormone levels. Our results highlight the ventromedial hypothalamus as an important center of behavioral differences across individuals and provide novel candidates for investigations into the regulation of individual variation in social behavior phenotype.

The effects of dopamine receptor 1 and 2 agonists and antagonists on sexual and aggressive behaviors in male green anoles.

The propensity to exhibit social behaviors during interactions with same-sex and opposite-sex conspecifics is modulated by various neurotransmitters, including dopamine. Dopamine is a conserved neurotransmitter among vertebrates and dopaminergic receptors are also highly conserved among taxa. Activation of D1 and D2 dopamine receptor subtypes has been shown to modulate social behaviors, especially in mammalian and avian studies. However, the specific behavioral functions of these receptors vary across taxa. In reptiles there have been few studies examining the relationship between dopaminergic receptors and social behaviors. We therefore examined the effects of D1 and D2 agonists and antagonists on sexual and aggressive behaviors in the male green anole lizard (Anolis carolinensis). Treatment with high doses of both D1 and D2 agonists was found to impair both sexual and aggressive behaviors. However, the D1 agonist treatment was also found to impair motor function, suggesting that those effects were likely nonspecific. Lower doses of both agonists and antagonists failed to affect social behaviors. These findings provide some evidence for D2 receptor regulation of social behaviors, but in contrast with previous research, these effects are all inhibitory and no effects were found for manipulations of D1 receptors. A potential reason for the lack of more widespread effects on social behaviors using moderate or low drug doses is that systemic injection of drugs resulted in effects throughout the whole brain, thus affecting counteracting circuits which negated one another, making measurable changes in behavioral output difficult to detect. Future studies should administer drugs directly into brain regions known to regulate sexual and aggressive behaviors.

Differences in forebrain androgen receptor expression in winners and losers of male anole aggressive interactions

Size matched male green anoles (Anolis carolinensis) were paired in a neutral setting and allowed to engage in aggressive displays. Winners and losers were apparent in each pair within 90min, resulting in stable dominant/subordinate dyads. Androgen receptor (AR) expression was assessed at three time points after the initial pairing, 2h, 3 days, and 10 days in dominants, subordinates, and two groups of control males housed alone or with a female for an equal period of time. Expression was quantified in three forebrain areas that have been implicated in aggression and reproductive social behavior in this species, the preoptic area (POA), the anterior hypothalamus (AH), septal area (SEP), and ventromedial nucleus of the posterior division of the dorsal ventricular ridge (PDVRVM ). There were significant overall group differences in AR mRNA expression in the POA and AH that appeared to result from higher POA AR expression in dominant males compared to other groups, and generally lower AR expression in subordinate males. Pairwise comparison revealed that dominants′ AR mRNA expression in the POA was significantly higher in the 2h and 3 day groups compared to that of subordinates, with a similar, but nonsignificant, difference in the 10 day group. Dominants had significantly higher AR mRNA expression in the AH compared to that of subordinates in the 2h group, but differences were not significant at later times. The results suggest that POA and AH sensitivity to androgens is increased in dominants compared to subordinates, and that the difference can be seen soon after the agonistic interaction establishing winners and losers.

An experimental test of the effect of signal size and performance capacity on dominance in the green anole lizard

Summary1. Many animals use signals to resolve disputes over resources. Some signals act as reliable indicators of other traits, such as whole‐organism performance or body condition, which may also be important for resolving disputes. Because of the correlations inherent in reliable signals, it is challenging to determine which variables are directly relevant for resolving aggressive interactions.2. We examined the relationships among dewlap size, bite force and condition, all traits that may be important to conflict resolution in male green anole lizards (Anolis carolinensis). Using a large sample of wild‐caught animals, we showed significant positive correlations between dewlap size and maximum bite force capacity, when each trait is corrected for its correlation with body size.3. We tested the relative importance of each trait to the outcome of interactions in a subset of our sample. We staged dominance encounters between size‐matched male green anoles after surgically reducing the dewlap size of one competitor.4. We show that reducing the size of the dewlap does not significantly change the outcome of staged interactions. Rather, males with higher values of bite force capacity were more likely to win fights. We hypothesize that during close‐proximity aggressive interactions, male green anoles use more direct means of assessing one another and that dewlap size functions as a signal of bite force primarily during long‐distance territorial displays. Body condition was correlated with bite force, but did not differ significantly between winners and losers.5. Our results show how an experimental approach can decouple reliable signals from their correlated traits to test which factors influence male contest resolution.

Experimental Test of Predation and Competition Pressures on the Green Anole (Anolis carolinensis) in Varying Structural Habitats

The Green Anole, Anolis carolinensis, has been widely studied in terms of its behavioral ecology. However, few studies have simultaneously compared the relative strengths of predation and male competition across temporal and spatial scales in Green Anoles, or other reptile species. We took an indirect experimental approach to compare these forces in Green Anoles by using attacks on clay models as a proxy for the relative intensity of predation and male competition. We measured the proportion of attacks on clay models in two divergent Green Anole populations with distinct habitats (discrete and continuous vegetation) across three seasons (fall, winter, and spring) and found strong evidence for spatial and temporal variation in the relative intensity of each component of selection. The frequency of bites from male Green Anoles was generally higher at an urban locality, whereas the frequency of bites from nonlizards was higher at a natural swamp locality. We also detected temporal variation in the intensity of these processes at both locations, with the intensity of male competition and predation changing in a concomitant fashion, the most intense periods occurring during the fall and spring months and the least intense during the winter months. Our results imply that mortality within some seasonal reptile species is composed of periods of relative calm punctuated by brief but intense “one-two punch” periods of selection.

Effects of season, testosterone and female exposure on c- fos expression in the preoptic area and amygdala of male green anoles

Expression of the immediate early gene, c- fos, was used to investigate changes in neuronal activity in forebrain regions involved in male sexual behavior following social, hormonal and/or seasonal manipulations in the male green anole. These factors all influence behavior, yet it is unclear how they interact to modify neuronal activity in forebrain regions, including the preoptic area (POA) and ventromedial nucleus of the amygdala (AMY). These regions are involved in the display of sexual behaviors in male green anoles as in many other vertebrates. To determine the effects of seasonal, hormonal and social cues on these brain areas, we investigated c- fos under environmental conditions typical of the breeding or non-breeding season in adult male green anoles that were castrated and implanted with either testosterone (T) or blank (Bl) capsules. We also manipulated social cues by exposing only half of the animals in each group to females. T enhanced courtship and copulatory behaviors, but decreased c- fos expression in the AMY. A similar, although not statistically significant, pattern was observed in the POA, and the density of c- fos+ cells was negatively correlated in that region with the number of extensions of a throat fan (dewlap) used during courtship. Therefore, it appears that in the male green anole, T may diminish c- fos expression (likely in inhibitory neurons) in the POA and AMY to create a permissive environment in which the appropriate behavioral response can be displayed.

Hormones, sexual signals, and performance of green anole lizards ( Anolis carolinensis)

The evolutionary processes that result in reliable links between male signals and fighting capacity have received a great deal of attention, but the proximate mechanisms underlying such connections remain understudied. We studied a large sample of male green anole lizards ( Anolis carolinensis) to determine whether testosterone or corticosterone predicted dewlap size and/or bite-force capacity, as dewlap size is known to be a reliable predictor of bite-force capacity in territorial males. We also examined whether these relationships were consistent between previously described body size classes (“lightweights” and “heavyweights”). Heavyweights had 50% higher testosterone concentrations than lightweights during the breeding season, suggesting a mechanism for the disproportionately larger heads and dewlaps and higher bite-forces of heavyweights. Plasma testosterone concentrations were positively correlated with dewlap size and bite-force performance in lightweights (but not heavyweights) but only because of mutual intercorrelation of all three variables with body size. We suggest two possibilities for the relationship between testosterone levels and body size: (1) testosterone promotes growth in this species or (2) smaller sexually mature males are unable to compete with larger males such that the benefits of elevated testosterone do not outweigh the costs. Corticosterone levels did not differ between the male morphs, and lightweights, but not heavyweights, showed an inverse relationship between testosterone levels and corticosterone levels. Our results suggest that testosterone is important for traits related to dominance in adult male green anoles and may influence the ability to compete with rivals via fighting ability or through the use of signals.

Androgen receptor expression and morphology of forebrain and neuromuscular systems in male green anoles displaying individual differences in sexual behavior

Investigating individual differences in sexual performance in unmanipulated males is important for understanding natural relationships between behavior and morphology, and the mechanisms regulating them. Among male green anole lizards, some court and copulate frequently (studs) and others do not (duds). To evaluate potential factors underlying differences in the level of these behaviors, morphology and androgen receptor expression in neuromuscular courtship and copulatory structures, as well as in the preoptic area and amygdala, were compared in males displaying varying degrees of sexual function. This study revealed that individual differences in behavior among unmanipulated males, in particular the extension of a throat fan (dewlap) used during courtship, were positively correlated with the size of fibers in the associated muscle and with soma size in the amygdala. The physiological response to testosterone, as indicated by the height of cells in an androgen-sensitive portion of the kidney, was also correlated with male sexual behavior, and predicted it better than plasma androgen levels. Androgen receptor expression was not related to the display of courtship or copulation in any of the tissues examined. The present data indicate that higher levels of male courtship behavior result in (or are the result of) enhanced courtship muscle and amygdala morphology, and that androgen-sensitive tissue in studs may be more responsive to testosterone than duds. However, some mechanism(s) other than androgen receptor expression likely confer this difference in responsiveness.

Courtship and copulation in the adult male green anole: Effects of season, hormone and female contact on reproductive behavior and morphology

Interactions among reproductive season, testosterone (T) and female presence were investigated on the structure and function of forebrain and neuromuscular systems controlling courtship and copulation in the green anole lizard. Under breeding (BS) or non-breeding (NBS) environmental conditions, male green anoles were implanted with either T or blank capsules and exposed to one of three female stimulus conditions: physical, visual or no female contact. T and at least visual exposure to females increased courtship displays (extension of a throat fan, or dewlap), and these effects were greater during the BS than NBS. T also facilitated copulation, and did so to a greater extent in the BS. The hormone increased soma size in the preoptic area (POA) and amygdala (AMY), and in the AMY the effects were greater in the BS than NBS. Cross-sectional areas of copulatory organs and associated muscle fibers were enhanced by T, and more so in the BS than NBS. However, no effects on morphology of dewlap motoneurons or muscles or copulatory motoneurons were detected. Thus, (1) changes in behavior and neural and/or muscular morphology are not always parallel and (2) differences in responsiveness to T exist across seasons and among tissues.

Aggressive experience alters place preference in green anole lizards, Anolis carolinensis

Males of many species establish territories from which they exclude conspecifics, and experience can influence the defence and stability of territorial boundaries. We used a conditioned place preference (CPP) procedure to determine whether engaging in a form of behaviour associated with territorial defence, aggressive behaviour, was rewarding for male green anoles and whether aggressive experience could alter the spatial/contextual preferences of these territorial lizards. When approached by a male conspecific, or when exposed to their reflection in a mirror, many male anoles show species-typical aggressive behaviours. We repeatedly exposed male anoles to a mirror or the nonreflective back of the mirror in opposing sides of a CPP apparatus that consisted of two contextually distinct chambers connected by a central tunnel. Control animals (N ¼ 10) were exposed to the back of the mirror in both sides of the apparatus. Following conditioning, animals that showed aggressive behaviour (N ¼ 13) when exposed to the mirror during conditioning increased the time that they spent in the side of the apparatus paired with the mirror relative to baseline. In contrast, the preferences of nonaggressive (N ¼ 8) and control animals were unchanged. These findings suggest that engaging in aggressive behaviour (and not simply seeing a conspecific) is rewarding for male green anoles and that aggression-related reward and associative learning could influence the formation and/or maintenance of stable territories.

Effects of Sarcophagid Fly Infestations on Green Anole Lizards (Anolis carolinensis): An Analysis across Seasons and Age/Sex Classes

A largely unresolved ecological issue concerns why normally rare parasites become common within reptile and amphibian populations. We gathered demographic data on incidence of infection and demographic consequences of a normally rare sarcophagid fly parasite (Lepidodexia blakeae) on a population of Green Anole Lizards (Anolis carolinensis). These sarcophagid flies primarily larviposit on adult male Green Anoles during the winter. Sampling by age and sex classes during two years (2003 and 2004) revealed that adult male anoles were infected at an unusually high rate (up to 16%) compared to other populations (only a few documented cases over the past 60 years) and relative to conspecific juveniles and females. Male anoles at Tulane exhibited high mortality compared to other nonparasitized populations, suggesting that parasitism negatively affects survivorship. Although the underlying cause of the high parasitism in the Tulane population remains unclear, several lines of evidence suggest that high and stressful levels of male competition may be a key factor.

Testosterone Regulates Androgen Receptor Immunoreactivity in the Copulatory, but not Courtship, Neuromuscular System in Adult Male Green Anoles

Androgens regulate the expression of male reproductive behaviour in diverse vertebrate species, often acting on androgen receptors (AR) to induce structural or functional changes in the nervous system and periphery. Male green anoles possess two sexually dimorphic neuromuscular systems, one controlling throat fan (dewlap) extension, which occurs during courtship, and the other mediating copulatory organ function. Although androgens are required for behavioural activation in both systems, testosterone has differential effects on the neuromuscular morphology. It increases the size of copulatory muscle fibres during the breeding season, but significant effects on dewlap muscle fibre size and motoneurone soma size in either system have not been detected. Corresponding to the lack of testosterone-induced morphological effects in the courtship system, relatively low levels of AR are expressed in the associated motoneurones. The present experiment had two goals, aiming to determine whether: (i) the other courtship and copulatory neuromuscular tissues express AR and (ii) testosterone and/or seasonal environmental changes regulate AR expression. The percentage of AR+ nuclei was evaluated in both the breeding and nonbreeding seasons in gonadally intact adult males (Experiment 1) and in castrated males treated with either testosterone or vehicle (Experiment 2). AR was extensively expressed in the dewlap and copulatory muscles, and in a high percentage of the copulatory motoneurones, but immunoreactivity did not vary across season. Testosterone increased the percentage of AR+ nuclei in the copulatory muscles of both breeding and nonbreeding males but not in the dewlap muscle or copulatory motoneurones. Finally, the target structures for both systems (cartilages and hemipenes) expressed AR in all animals. Therefore, the effects of testosterone on AR immunoreactivity suggest that up-regulation of the receptors may be important for morphological change. However, because all structures investigated in the present experiment expressed AR, the data also indicate that the receptors are involved with other functions.

Opponent recognition in male green anoles, Anolis carolinensis

Male green anoles convey territorial ownership to rivals through use of stereotyped aggressive displays, with intrasexual interactions often escalating to physical combat. In the laboratory, losers of such interactions significantly reduce their aggressive behaviour when interacting with the same opponent 7 days later. In the current study, we examined whether this behavioural modification is a function of opponent recognition. Pairs of size-matched male A. carolinensis were allowed to interact for 2 h. Following this, subjects were separated for varying intervals, and then participated in a second interaction with either the same or an unfamiliar opponent. Losers of the first interaction significantly reduced their total aggressive behaviour when re-paired with the same opponent after a 3-day interval but not after a 10-day interval between the first and second interaction. However, losers of the first interaction failed to show reduced aggression during their second interaction when paired 3 or 10 days later with an unfamiliar opponent. Therefore, A. carolinensis appear to learn information specific to their opponent during an initial agonistic interaction, with the salience of this information declining when the interval between interactions increases. Males also showed low levels of chemosensory taste behaviour during agonistic interactions, suggesting that opponent recognition does not rely on chemoreception. Similarities between our results and findings from other vertebrate species suggest that the motivation to behave aggressively during social interactions can be influenced by opponent recognition rather than by previous experience of social defeat alone.

Androgen Metabolism in the Brain of the Green Anole Lizard (Anolis carolinensis): Effects of Sex and Season

Courtship behavior in male green anoles is partly mediated by the 5α-reductase enzyme, which converts testosterone (T) to 5α-dihydrotestosterone. This study aimed to determine whether the activity of 5α-reductase is enhanced in breeding males compared to females and nonbreeding males who do not normally display masculine behaviors. In some cases, aromatase, which converts T to 17β-estradiol, also was assessed to determine whether the pattern of its activity in anoles is similar to that in other vertebrates. 5α-Reductase is greatest in the brainstem, so its activity was determined separately in homogenates of whole brain and brainstems. The following comparisons were done in different assays: (1) breeding males with breeding females, (2) nonbreeding males with nonbreeding females, and (3) breeding males with nonbreeding males. Aromatase activity was greater in breeding males (mean ± SE, 0.61 ± 0.06 fmol/min/mg protein) than in breeding females (0.41 ± 0.08 fmol/min/mg protein). It was also greater in breeding males (0.84 ± 0.16 fmol/min/mg protein) than in nonbreeding males (0.33 ± 0.07 fmol/min/mg protein). In contrast, sex or seasonal differences did not exist in 5α-reductase activity. The results are consistent with those of other vertebrate species in which male-biased sex dimorphisms and seasonal differences occur in aromatase, but not in 5α-reductase activity. The greater levels of aromatase activity in breeding male anoles suggest that this enzyme might mediate male-specific functions. The equivalently high levels of 5α-reductase activity in both sexes suggests that, in addition to facilitating male courtship behavior, the enzyme has a basic function common to both sexes.

Development of a Sexually Dimorphic Neuromuscular System Involved in Green Anole Courtship Behavior

During courtship and aggression, adult male green anoles (Anolis carolinensis) extend a large red throat fan called a dewlap. Adult females have a very small dewlap; they rarely use it during agonistic encounters and never during courtship. The motoneurons, nerve, muscle fibers, neuromuscular junctions, and cartilage pieces responsible for dewlap extension are all larger in adult males than females, parallel to the behavioral dimorphisms. However, the general developmental pattern of these structures, and when they become sexually dimorphic, was largely unknown. In the present study, we examined the length of the 2nd ceratobranchial cartilages (which unfold the dewlap), muscle fiber size, nerve cross-sectional area, and motoneuron soma size, as well as measures of body size (snout-vent length and body weight) at post-hatching days 1, 30, 60, 75, and 90. All measures other than motoneuron soma size increased over time. However, only cartilage length and muscle fiber size became sexually dimorphic in the first three months after hatching. The cartilage was greater in males than females by d60, and muscle fiber size differentiated by d75. The results indicate that peripheral structures involved in dewlap extension differentiate prior to those in the brain and thus, because males and females in this age range are identical in size, dewlap (cartilage) size could be a relatively specific factor contributing to the sexual differentiation of the rest of the neuromuscular system.

Sexual Dimorphisms in the Brainstem of the Green Anole Lizard

Male green anoles (Anolis carolinensis) court females by extending a bright red throat fan, called a dewlap. The dewlap is larger in males than in females who do not display the structure in the context of courtship. The ceratohyoid muscle and second ceratobranchial cartilage which control dewlap extension are significantly larger in male than in female anoles. Neurons innervating the ceratohyoid muscle were retrogradely labeled by injecting the muscle with fast blue and were found to be located in the vagal portion of nucleus ambiguus (Amb X), as well as in the region containing the glossopharyngeal portion of nucleus ambiguus (Amb IX) and the ventral portion of the motor nucleus of the facial nerve (nVIImv). Cells in both areas were larger in males than in females. These parallels between structure and function provide the opportunity to study mechanisms of sexual differentiation of brain and behavior.

Territorial Defense by Male Green Anoles: An Experimental Test of the Roles of Residency and Resource Quality

To test the hypothesis that males will more vigorously defend high-value territories (those containing females versus those with no females), male green anoles (Anolis carolinensis) were allowed to establish residency in 50 x 25 x 31-cm terraria with or without a female present. Each male was then removed for 1, 3, 5, or 7 h while an intruder was given access to the terrarium. The original owner was then returned to the terrarium with the intruder present, and the aggressive acts of both males were recorded. An aggression index was calculated for each owner-intruder pair to determine which member the contest. Owners won more contests than intruders after all removal times except 7 h. Aggression did not increase significantly at intermediate removal times, when both males may have perceived themselves as territory owners. When the value of a territory was increased by the presence of a female, aggression by both the owner and the intruder increased significantly after the 3-h owner removal time, and aggression by the intruder (but not owner) increased significantly after the 5-h owner removal time. Winners of territorial contests in male green anoles seem to be determined by the resource holding potential of the contestants, the value of a contested territory, and particularly the ability of males to assess each other.

Contextual signalling and the structure of dyadic encounters in Anolis carolinensis

Abstract. During conflicts over resources (contests), differences between opponents (asymmetries), which can result in settlement, may be revealed through behaviour patterns constrained by static variables such as body mass (assessment signals), or by labile, potentially dishonest behaviour (contextual signals). Little is known about how contextual signals may settle complex contests. This study investigates how the effects of pre-contest social treatments on labile aspects of internal states influence contest behaviour in male green anoles, Anolis carolinensis. Lizards were subjected to one of two pre-contest treatments which manipulated levels of internal factors that influence the propensity to win contests. Low-level lizards were residents for half an hour in a contest arena. High-level lizards were residents for 20-24 h with a subordinate male and a female present. Comparisons were subsequently made between three contest types: low level; high level and asymmetric. Contests were staged by removing an opaque partition separating two residents. Each contest type generated different behaviour patterns, and the frequency with which these behaviour patterns were displayed appeared to be particularly important in separating contest types. In asymmetric contests, high-level lizards won 20 of 23 contests, but the differences in treatment-level did not have detectable effects on behaviour. In all contests, many behaviour patterns of winners were correlated with those of losers. This suggests that opponents matched behaviour, while subtle interactions between opponents caused different contest-types to follow different courses. A lizard could become aware of an asymmetry by its opponents behaviour without a clear behavioural difference between opponents.

Geographic variation in the critical thermal minimum of the green anole, Anolis carolinensis (sauria:Iguanidae), along a latitudinal gradient

1. 1. Critical thermal minimum (CTMin) tests were conducted on adult male green anoles ( Anolis carolinensis) collected in central Florida, southern Georgia and eastern Tennessee. 2. 2. CTMin was determined on the afternoon of capture to obtain a temperature range from which a laboratory acclimation temperature could be extracted. After being held in the laboratory at 25 ± 2°C for 14–18 days, lizards were cold-acclimated to 9°C for 48 hr prior to CTMin retesting. 3. 3. The Tennessee population was significantly more cold resistant than the Florida and Georgia populations, suggesting a genetic basis for the variation noted.

Thermoperiodic and photoperiodic influences on daily and seasonal changes in the physiology of the male green anole, Anolis carolinensis

AbstractMale green anole lizards, Anolis carolinensis, were kept for 22 days on one of five temperature regimes: constant 20°C, constant 30°C, or a thermoperiod (8 h at 30°C, 16 h at 20°C) with heat onset at 0, 8, or 16 h after light onset. Groups of animals on each temperature regime were sacrificed at one of six times of day, every 4 h, for 24 h. The experiment was repeated at three different times of year: April (animals were kept on a photoperiod of LD 13:11), August (LD 13.5:10.5), and December (LD 10.5:13.5). Weight gain, testes weights, reproductive stage, and abdominal fat body weights varied with the temperature treatment and with the season. Depending on the time of day lizards received the warm temperature phase of the thermoperiod, increased heat could be stimulatory to weight gain and reproductive indexes in comparison to heat treatment at other times of day. The effect of constant temperatures on testis state and body weight gain was different from that of the thermoperiod regimes and of the natural environment on these processes.There were also diel variations in both testis DNA synthesis and fat body lipogenesis in the green anole. The photoperiod appeared to set the daily variation of DNA synthesis in the testis, whereas the thermoperiod phased the daily variation of fat body lipogenesis.