Steroid hormone mediation of limbic brain plasticity and aggression in free-living tree lizards, Urosaurus ornatus

The neural mechanisms by which steroid hormones regulate aggression are unclear. Although testosterone and its metabolites are involved in both the regulation of aggression and the maintenance of neural morphology, it is unknown whether these changes are functionally related. We addressed the hypothesis that parallel changes in steroid levels and brain volumes are involved in the regulation of adult aggression. We examined the relationships between seasonal hormone changes, aggressive behavior, and the volumes of limbic brain regions in free-living male and female tree lizards (Urosaurus ornatus). The brain nuclei that we examined included the lateral septum (LS), preoptic area (POA), amygdala (AMY), and ventromedial hypothalamus (VMH). We showed that the volumes of the POA and AMY in males and the POA in females vary with season. However, reproductive state (and thus hormonal state) was incompletely predictive of these seasonal changes in males and completely unrelated to changes in females. We also detected male-biased dimorphisms in volume of the POA, AMY, and a dorsolateral subnucleus of the VMH but did not detect a dimorphism between alternate male morphological phenotypes. Finally, we showed that circulating testosterone levels were higher in males exhibiting higher frequency and intensity of aggressive display to a conspecific, though brain nucleus volumes were unrelated to behavior. Our findings fail to support our hypothesis and suggest instead that plasma testosterone level covaries with aggression level and in a limited capacity with brain nucleus volumes but that these are largely unrelated relationships.     

Isolation and characterization of microsatellite markers in tree lizards (Urosaurus ornatus)

Tree lizards (Urosaurus ornatus) are a highly accessible species useful in testing theories of life-history evolution and behavioural ecology. A polymerase chain reaction-based method was used to isolate nine polymorphic tetranucleotide microsatellite loci from the genome of these lizards. The isolated loci displayed 5 to 9 alleles in the populations screened. Observed heterozygosity ranged from 0.333 to 0.900. Results indicate these loci now provide a basis to study mate choice, parentage, and population genetic structure within this species.     

Clinical efficacy of antiparasite treatments against intestinal helminths and haematic protozoa in Gallotia caesaris (lizards)

Intestinal helminths and blood protozoa are common parasites of lizards. In captivity they can be a serious problem, but no information on treatments is available. In this study, several antiparasitic drugs were studied in Gallotia caesaris (Lacertida) from La Gomera, the Canary Islands, Spain. A lack of efficacy of chloroquine, and a decrease in haemogregarine (Apicomplexa) infection by atovaquone-proguanil was demonstrated. Furthermore, a positive effect of fenbendazole against intestinal nematode infection was observed. In the Canary Islands, Gallotia bravoana and Gallotia simonyi (Lacertida) are included in a captive breading recovery plan. The present results are useful in order to keep these animals in good condition and to control their parasites. Moreover, these results are important for reptiles kept as pets, due to the previous lack of knowledge of parasite management.     

Aggression frequency and intensity, independent of testosterone levels, relate to neural activation within the dorsolateral subdivision of the ventromedial hypothalamus in the tree lizard Urosaurus ornatus

The mechanisms by which testosterone regulates aggression are unclear and may involve changes that alter the activity levels of one or more brain nuclei. We estimate neural activity by counting immunopositive cells against phosphorylated cyclic AMP response element binding protein (pCREB). We demonstrate increased pCREB immunoreactivity within the dorsolateral subdivision of the ventromedial hypothalamus (VMHdl) following an aggressive encounter in male tree lizards Urosaurus ornatus. This immunoreactivity is induced both by exposure to and performance of aggressive behaviors. This dual activation of the VMHdl suggests its possible role as an integration center for assessment and expression of aggressive behavior. Furthermore, pCREB induction was greater in encounters involving higher frequency and intensity of aggressive display, demonstrating a direct relationship between neural activation and behavior. The VMHdl is also rich in steroid receptors. In a second experiment involving hormone manipulations, testosterone treatment increased aggression levels, though it did not increase the number of pCREB positive cells within the VMHdl. This lack of an effect of testosterone on pCREB induction within the VMHdl may be due to induction arising from the behaviors of conspecifics (especially in low-testosterone, low-aggression individuals), variation in aggression mediated by other variables, or regulation of aggression by circuits outside of the VMHdl. Together, these findings support a notion of the VMHdl as a nucleus involved in integrating afferent and efferent information within the neural aggression-control circuit.     

Social modulation of testosterone levels in male black howlers (Alouatta pigra)

The influence of social factors on the modulation of male testosterone levels has been demonstrated among several vertebrate species. In addition to sexual activity, parental care and reproductive competition affect testosterone secretion. We examined variations in testosterone levels among male black howlers (Alouatta pigra) in various social contexts. Fecal samples were collected from nine males living in five different groups in the Mexican state of Campeche. The potential for intragroup and extragroup competition varied among the groups. The number of resident males living in the groups was the only variable that significantly explained variations in testosterone levels. Males living in unimale groups had higher testosterone levels; the highest testosterone levels were recorded for males that had experienced a shift from multimale to unimale group compositions. In this species, the probability of being challenged by extragroup males and evicted from the group during immigration events increases when males live in unimale groups. Therefore, our results suggest that male black howlers respond to competition for group membership by increasing their testosterone levels. In this context, testosterone secretion represents an anticipatory response to reproductive conflicts. Therefore, although males living in unimale groups have exclusive access to females, they face higher physiological costs associated with sustaining high testosterone levels for extended time periods.     

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.     

Acute corticosterone elevation enhances antipredator behaviors in male tree lizard morphs

In response to stressful events, most vertebrates rapidly elevate plasma glucocorticoid levels. Corticosterone release stimulates physiological and behavioral responses that can promote survival while suppressing behaviors that are not crucial to immediate survival. Corticosterone also has preparatory effects for subsequent stressors. Using male tree lizards (Urosaurus ornatus), we tested our prediction that elevated corticosterone is important for mediating and enhancing antipredator behaviors. Male tree lizards express developmentally fixed polymorphisms that are mediated by early organizational actions of steroid hormones, and thus we also tested the hypothesis that morph-specific differences in antipredator behaviors of adults are independent of circulating corticosterone levels. Plasma corticosterone levels were elevated exogenously for 12-16 h using non-invasive dermal patches, and we then compared the behavioral responses of these corticosterone-patched males to control-patched males during a simulated encounter with a caged predator (collared lizard, Crotaphytus nebrius) in outdoor enclosures. Elevating corticosterone did not alter the antipredator behavioral repertoire of each male morph, but did enhance their responses during the predator encounter: all corticosterone-patched males responded more quickly, hid longer, and displayed more toward the predator than control-patched males. With the corticosterone patch, the non-territorial and wary orange morph was still behaviorally the most wary morph, responding more quickly and hiding longer than either the bolder orange-blue or mottled morphs. Smaller males were generally warier than larger males, regardless of the endocrine treatment or color morph type. In sum, elevated circulating corticosterone enhances antipredator responses for all male tree lizard morphs, without altering morph-specific or size-specific differences in their behavioral responses.     

Manipulation of egg production reveals costs of reproduction in the tree lizard (Urosaurus ornatus)

Life-history theory predicts that the allocation of energy to current reproduction is associated with a decrement in future fecundity, future survival, or both. I treated this notion as the cost hypothesis, and tested the assumption that current reproduction exacts a cost in future survival and fecundity. Surgical manipulations of egg production were applied to natural populations of the tree lizard, Urosaurus ornatus, in south western New Mexico by yolkectomy surgery in two different years. I reduced the number of eggs produced in the first clutch during vitellogenesis by approximately 50% in yolkectomized females relative to controls. Subsequent survival, fecundity, and growth of females were followed for two or three years, depending on the cohort. Treated females in both cohorts showed significantly higher growth and survivorship throughout the experiment than in controls. After 2 years, yolkectomized females had grown an additional 2 mm (snout-vent length) compared to controls, enough for them to add on average an additional egg to their next clutch. This demonstrated a cost in terms of future fecundity through a reduction in growth and an increase in mortality in these lizards.     

Performance and thermal sensitivity of the southernmost lizards in the world, Liolaemus sarmientoi and Liolaemus magellanicus

Locomotor activity performance of reptiles is largely temperature dependent and, in harsh environments with short activity periods during the day and throughout the year, plays a vital role in the fitness of the species. This particular study focuses on the performance and the thermal sensitivity for running, at different body temperatures, of the two southernmost species of lizards in the world, Liolaemus sarmientoi and Liolaemus magellanicus, studied at two locations in the south of Santa Cruz province, Argentina (51°S, 70°W and 50°S, 72°W; 133 m asl). The speed of sprint and long runs was measured in the field to determine the physiological performance of lizards at different air temperatures. In both species speed increases with the temperature, and they reach the highest performance at high temperatures. The difference between activity and thermal optima suggests that L. magellanicus has colonized its actual environment recently, and that it has not had enough time for its physiological mechanisms to evolve and achieve a maximum performance at the cold temperatures they have to tolerate at present. In contrast, L. sarmientoi achieved a high performance over a wider range of temperatures that included temperatures lower than the preferred temperatures in the lab, which they can generally find in their environment.     

Corticosterone, locomotor performance, and metabolism in side-blotched lizards (Uta stansburiana)

Elevated levels of circulating corticosterone commonly occur in response to stressors in wild vertebrates. A rise in corticosterone, usually in animals of subordinate rank, results in a variety of effects on behavior and physiology. Behavioral and physiological responses to short-term increases in corticosterone are well studied. In contrast, the effects of chronic elevated levels of corticosterone are poorly understood, particularly in lizards. Here, we examined the long-term effects of exogenous corticosterone on locomotor performance, resting and active metabolic rate, and hematocrit in male side-blotched lizards Uta stansburiana. Corticosterone implantation resulted in higher levels of stamina relative to sham-surgery controls. In addition, lizards with elevated corticosterone exhibited lower resting metabolic rates relative to controls. Corticosterone had no effect on peak activity metabolism but did result in faster recovery times following exhaustive exercise. We suggest that elevated levels of corticosterone in response to dominance interactions promote enhanced locomotor abilities, perhaps as a flight response to avoid agonistic interactions. Furthermore, stressed lizards are characterized by lower resting metabolic rates, which may serve as strategy to conserve energy stores and enhance survival.     

Hormonal correlates of dominance in meerkats (Suricata suricatta)

In cooperatively breeding meerkats (Suricata suricatta), individuals typically live in extended family groups in which the dominant male and female are the primary reproductives, while their offspring delay dispersal, seldom breed, and contribute to the care of subsequent litters. Here we investigate hormonal differences between dominants and subordinates by comparing plasma levels of luteinizing hormone (LH), estradiol and cortisol in females, and testosterone and cortisol in males, while controlling for potential confounding factors. In both sexes, hormone levels are correlated with age. In females, levels of sex hormone also vary with body weight and access to unrelated breeding partners in the same group: subordinates in groups containing unrelated males have higher levels of LH and estradiol than those in groups containing related males only. When these effects are controlled, there are no rank-related differences in circulating levels of LH among females or testosterone among males. However, dominant females show higher levels of circulating estradiol than subordinates. Dominant males and females also have significantly higher cortisol levels than subordinates. Hence, we found no evidence that the lower levels of plasma estradiol in subordinate females were associated with high levels of glucocorticoids. These results indicate that future studies need to control for the potentially confounding effects of age, body weight, and access to unrelated breeding partners before concluding that there are fundamental physiological differences between dominant and subordinate group members.     

Effects on the thermoregulatory efficiency of two native lizards as a consequence of the habitat modification by the introduction of the exotic tree Acacia longifolia

Habitat modification alters several aspects of the original fauna, among them the opportunity for thermoregulation. Here, we studied the thermal biology of sympatric populations of two lizard species (Liolaemus multimaculatus and Liolaemus wiegmannii) in two different situations; a grassland without trees (natural habitat) and in a grassland plus the exotic tree Acacia longifolia (modified habitat), aiming to assess whether the structural alteration of native Pampean coastal grasslands of Argentina affects the thermal biology of these lizards. Field body temperatures, laboratory preferred temperatures, micro-environmental temperatures, operative temperatures, thermoregulatory efficiency and spatial distribution of each species were analyzed in both habitats. Environmental operative temperature was 0.64 °C lower in the modified habitat (Te=38.39 °C) than in the natural (Te=39.03 °C). Thermoregulatory efficiency (E) of L. wiegmannii was lower in modified sites (E=0.58) than in natural sites (E=0.70). This difference may be because this lizard occupied shaded microhabitats under acacias, with suboptimal thermal features. In contrast, L. multimaculatus in the modified habitat restricted its activity to open microenvironments that retained a similar structure to that of the native habitat, while maintaining high thermoregulatory efficiency in both habitat types (E_modified=0.92; E_natural=0.96). Although these two lizard species are phylogenetically close, they respond differently to human-induced changes in their thermal environments. The introduction of A. longifolia into coastal grasslands for L. wiegmannii in particular, this introduction converts its native habitat into a suboptimal thermal environment.     

Increased sibling competition does not increase testosterone or corticosterone levels in nestlings of the spotless starling (Sturnus unicolor)

Nestling begging in passerine birds is a complex behaviour that is shaped by a multitude of ecological factors and could be physiologically mediated by varying levels of steroid hormones. Previous research has shown links between sibling competition and testosterone and corticosterone in several bird species. The spotless starling (Sturnus unicolor) is a medium sized passerine in which nestlings compete intensively for resources, often resulting in marked size hierarchies that can have profound effects on their fitness. We tested the hypothesis that an increase in sibling competition levels would result in increases in testosterone and corticosterone in this species. To this end we conducted a brood size manipulation, creating small, medium and large broods. This manipulation had the expected effect on morphology: nestling size and mass decreased with increasing brood size. Androgen levels varied in response to brood size manipulation but, contrary to expectations, the largest concentrations were found in reduced brood sizes. Corticosterone levels increased with increasing brood size, but this effect disappeared when we corrected for the time taken to process nestlings. Cell-mediated immune response was found to decrease with increasing brood size and testosterone levels. The results suggest that the proposed link between testosterone and corticosterone and sibling competition does not hold in this species, and underlines the diversity of species-specific responsiveness to steroids.     

Spilanthes acmella ethanolic flower extract: LC-MS alkylamide profiling and its effects on sexual behavior in male rats

According to Indian Systems of Medicine, Spilanthes acmella (L.) Murr. (Family - Asteraceae), is considered effective in the treatment of sexual deficiencies especially due to ageing. In the present study, characterization of ethanolic extracts of the Spilanthes acmella flower and its effect on general mating pattern, penile erection and serum hormone levels of normal male Wistar albino rats were investigated and compared with sildenafil citrate. In vitro nitric oxide release was also investigated in human corpus cavernosum cell line. As N-alkylamides are a promising group, their profiling was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS¹ and MS² fragmentation data were used for identification purposes. For assessment of sexual behavior, animals were divided into five groups of eight male rats. The extracts (50, 100 and 150 mg/kg body weight/day) and sildenafil citrate (5 mg/kg body weight/day) (positive control) were administered orally for 28 days. The behavioral and sexual parameters were observed at days 0, 15, 28 and after a lapse of 7 and 14 days of discontinuance of drug treatment. Five N-isobutylamides, one 2-methylbutylamide and one 2-phenylethylamide were identified. The orally administered extract had a dose dependent positive effect on mounting frequency, intromission frequency and ejaculation frequency and the most significant effects (p < 0.05) were observed at 150 mg/kg treatment, even after a lapse of 7 and 14 days of discontinuance of drug treatment. A dose dependent effect was also observed on the FSH, LH and testosterone serum levels. With 150 mg/kg of ethanolic extract the values for FSH, LH and testosterone were 3.10 ± 0.25 mlU/ml, 6.87 ± 0.18 mlU/ml and 3.72 ± 0.12 ng/ml, respectively. In vitro nitric oxide release was 21.7 ± 2.9 μM, which was significantly higher compared to the control group (p < 0.01). Sildenafil citrate exhibited also a significant effect on NO release, but no effect on hormone levels of rats was observed. The aphrodisiac potential of an ethanolic Spilanthes acmella extract was demonstrated in vitro and in vivo. N-Alkylamides might attribute to the improved sexual potential. Study lends support to the traditional utilization of S. acmella as a sexual stimulating agent.     

Structural alterations in the male reproductive system of the freshwater crayfish, Cherax quadricarinatus (Decapoda, Parastacidae)

No diseases affecting reproductive performance have been previously reported in freshwater crayfishes. This study aims to characterise one reproductive system abnormality found in males of Cherax quadricarinatus reared in captivity. Fifteen adult males of C. quadricarinatus (70-110 g) were purchased from San Mateo S.A. farm (Entre Ríos, Argentina) each season during 2007. Macroscopic analysis showed that 26.6% of the animals sacrificed in winter presented brownish distal vasa deferentia. Histological analysis showed different levels of structural abnormality in the epithelium of the vasa deferentia and spermatophore. Granular and hyaline haemocytes were identified within the vasa deferentia but no significant differences were found in the sperm count between normal and brownish vas deferens. Histological analysis of the crayfishes sacrificed in autumn also showed these modifications in 22% of the animals, however, they did not show the brownish colour under macroscopic analysis. The similarities between the male reproductive system syndrome in shrimps and the abnormalities found in C. quadricarinatus are notable. An unspecific response to thermic stress is a possible explanation of these structural alterations.     

Effects of different male remating intervals on the reproductive success of Choristoneura rosaceana males and females

The mass of the spermatophore transferred by a previously mated Choristoneura rosaceana male increases with time elapsed since the last mating but, even after 4 days, it never reaches the mass of the spermatophore of a virgin male. However, spermatophore mass is clearly not a good indicator of the male reproductive investment as the quantity of sperm in the second ejaculate of a previously mated male is the same as that of his first, if he is allowed a 2 (eupyrene sperm) to 3 day (apyrene sperm) recovery period. The interval between the first two matings had no influence on female fecundity or longevity but significantly affected fertility if the male had only 1 day to recover. The length of the post-copulatory refractory period was also shorter in females mated with previously mated males than in those mated with virgins, regardless of the male's remating interval. Furthermore, a significant variation in the eupyrene sperm content of the spermatophore transferred by virgin males had no influence on the length of the female refractory period. Globally, these results support the hypothesis that a factor, other than sperm numbers in the spermatheca, is responsible for maintaining the inhibition of pheromone production in this species.     

Variability in response of four populations of Amblyseius largoensis (Acari: Phytoseiidae) to Raoiella indica (Acari: Tenuipalpidae) and Tetranychus gloveri (Acari: Tetranychidae) eggs and larvae

Raoiella indica (Acari: Tenuipalpidae) is a phytophagous mite that recently invaded the Neotropical region. A predatory mite Amblyseius largoensis (Acari: Phytoseiidae) has been found associated with R. indica in Florida. This study evaluated A. largoensis by determining its likelihood of consuming eggs and larvae of R. indica and Tetranychus gloveri (Acari: Tetranychidae) under no-choice and choice conditions. To detect variations in the response of A. largoensis to R. indica, four populations of predators were examined: (1) predators reared exclusively on R. indica in the laboratory for 2 years, (2) predators reared on T. gloveri in the laboratory for 2 months but reared on R. indica for 2 years previously, (3) predators collected from a field infested with R. indica, and (4) predators collected from a field that had never been infested with R. indica. Results of this study suggest that A. largoensis is likely to accept and consume high numbers of R. indica eggs regardless of their previous feeding experience. In contrast, all populations consumed relatively fewer R. indica larvae than the other prey tested. Predators previously exposed to R. indica were more likely to consume R. indica larvae. By contrast, predators not previously exposed to R. indica showed the lowest likelihood of choosing to feed on this prey item. Plasticity in the response of A. largoensis to R. indica larvae could be associated to selection, learning, or a combination of both. The possible implications of the observed differences in terms of biological control of R. indica are discussed.     

Transformations of testosterone and related steroids by Botrytis cinerea

Botrytis cinerea (strain AM235) was used to investigate the transformations of testosterone and related steroids. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. Botrytis cinerea converts the examined substrates mainly to 7 alpha-hydroxy derivatives. 1-Dehydrotestosterone was also significantly hydroxylated at a 14 alpha-position.     

Experimentally elevated testosterone increases status signalling in male Greylag geese (<Emphasis Type="Italic">Anser anser</Emphasis>)

Testosterone modulates male vertebrates sexual and social behaviour. We experimentally investigated the testosterone-sensitive behaviours in male greylag geese (Anser anser) by implanting silastic tubes containing crystalline testosterone during the mating season (February; 5 implanted and 5 control males) and in the early winter (November; 7 and 7). Focal animals were part of a semi-tame, unrestrained flock with fully intact social relationships. Excreted testosterone and corticosterone immunoreactive metabolites (TM, BM) were determined by enzyme immunoassay. Individual faecal samples and behavioural protocols were collected daily over a period of 5 weeks, including 1 control week before implantation. In February, no significant behavioural effects of the supplemental testosterone were observed, which may be due to the naturally occurring high systemic androgen levels in spring. In November, however, implanted males had higher TM excretion rates and performed status signalling behaviour (beak up) more frequently than control males. No differences between implanted and control males were found with respect to BM, agonistic interactions or vigilance behaviour. Furthermore, during the second week after implantation, TM positively correlated with the frequency of beak up of implanted males, whilst their female partners were attacked with lower latency by other members of the flock than the females of control males. Hence, status signalling in greylag ganders seems to be testosterone-sensitive year-long and inappropriate status signalling of males may draw attacks towards their females.     

Testosterone increases display behaviors but does not stimulate growth of adult plumage in male golden-collared manakins (Manacus vitellinus)

In order to attract females, male golden-collared manakins gather in leks and perform a complex display consisting of acrobatics accompanied by loud "wingsnapping". During this display, males show off their yellow beard and yellow, black, and green plumage that is striking in comparison to the dull green plumage of young males and females. We investigated the role of testosterone (T) in activating the display of manakins and in stimulating the growth of the adult male plumage. T regulates song, copulation, and territorial aggression in temperate species. In tropical species, however, T levels can be relatively low year round, which has raised questions about the involvement of T in courtship display and male aggression in these species. In neither temperate nor tropical species has the role of hormones in the shift from juvenile to adult plumage been well studied. Therefore, we implanted green-plumaged birds and adult males with either a T pellet or an inert pellet (controls) and observed the display behaviors of these birds in the field and in captivity. In captive birds, we also plucked feathers from sexually dimorphic regions and observed color and regeneration rate of new feathers. We found that birds implanted with T increased several display behaviors compared to controls. All plucked feathers grew back the same color as prior to treatment; however, we observed some differences in feather growth rate between T-treated birds and controls.