Male Behavior Research Articles

A classic screening marker does not affect antennal electrophysiology but strongly regulates reproductive behaviours in Bactrocera dorsalis.

The key phenotype white eye (white) has been used for decades to selectively remove females before release in sterile insect technique programs and as an effective screening marker in genetic engineering. Bactrocera dorsalis is a representative tephritid pest causing damage to more than 150 fruit crops. Yet, the function of white in important biological processes remains unclear in B. dorsalis. In this study, the impacts of the white gene on electrophysiology and reproductive behaviour in B. dorsalis were tested. The results indicated that knocking out Bdwhite disrupted eye pigmentation in adults, consistent with previous reports. Bdwhite did not affect the antennal electrophysiology response to 63 chemical components with various structures. However, reproductive behaviours in both males and females were significantly reduced in Bdwhite-/- . Both pre-copulatory and copulation behaviours were significantly reduced in Bdwhite-/- , and the effect was male-specific. Mutant females significantly delayed their oviposition towards γ-octalactone, and the peak of oviposition behaviour towards orange juice was lost. These results show that Bdwhite might not be an ideal screening marker in functional gene research aiming to identify molecular targets for behaviour-modifying chemicals. Instead, owing to its strong effect on B. dorsalis sexual behaviours, the downstream genes regulated by Bdwhite or the genes from white-linked areas could be alternate molecular targets that promote the development of better behavioural modifying chemical-based pest management techniques.

Detrimental impact of a heatwave on male reproductive behaviour and fertility

Understanding how heatwaves impact on different aspects of mating behaviour and fertility is getting increasingly important. In this context, laboratory fertility and mating experiments involving manipulation and exposure of insects to different thermal conditions are common procedures. To conduct such experiments practical methods such as dyes are needed for an easy, non-invasive discrimination of individuals. We report here a study measuring the effect of an extended heat stress applied to males on several parameters of mating behaviour and fertility of laboratory populations of Drosophila subobscura derived from two distinct European locations. We found highly detrimental effects of heatwave on mating behaviour—with longer (courtship and copulation) latencies and lower mating occurrence but no changes in mating duration—and fertility, with reduced fecundity and reproductive success. Furthermore, we also tested the efficacy of food dye as a marker for individual discrimination and mating occurrence. While food dye did not allow to infer the occurrence of a mating based on a transfer of coloration from male to female, it did not affect mating and fertility, attesting its utility has a method for discriminating individuals within mating experiments in the context of thermal studies. Importantly, despite the fact that the heatwave was only applied in males, we observed an impact on behaviour of females that mated with stressed males, by often refusing their nuptial feeding. This opens possibilities for further integrated research on the changes of female and male mating behaviour and fertility under different thermal scenarios.

THE RESTRUCTURING OF GENDER MODELS IN THE NOVEL BY E.N. CHIRIKOV MY ROMANCE

The novel My Romance (Paris, 1929) written in exile by E.N. Chirikov (1864–1932) and dedicated to the so-called ‘tragedy of refugee’ has become the object of consideration in this article. In this novel, the writer started from thinking about the fate of millions of exiles who were forced to leave Russia as a result of the Civil War and the Red Terror, and focused on depicting the fate of a woman in conditions of loss of home, family and former social status. Chirikov’s appeal to the ‘women’s issue’ led to the use of a gender approach in the analysis of the text of the work, which in turn allowed to demonstrate the unique place of My Romance in the literature of the Russian emigration. The latter actually ignored the problem of changing of women’s status in the family circle and beyond provoked by the very course of history in the first quarter of the 20th century, and focused exclusively on preserving for posterity the memory of pre-revolutionary Russia, etc. Chirikov in My Romance clearly recorded the breaking of traditional female-male gender ‘roles’, revealing their dependence on socio-political processes. Thus, he discovered the artificiality and conventionality of many norms of female and male behavior familiar to contemporaries and summed up the debate about the status of women in the family circle and beyond which had been heard in Russian society for almost a century. At the same time, Chirikov demonstrated in My Romance that a man compared to a woman is more painfully enduring the loss of traditional gender functions, revealing greater spiritual weakness and moral vulnerability

Mating with dead conspecifics in an invasive ladybird is affected by male sexual fasting and time since the female’s death

High promiscuity and low mating partner choosiness in insects can sometimes result in a deviated mating behaviour such as mating with already dead individuals. In this study we investigated the occurrence of necrophilic behaviour in males of the invasive ladybird Harmonia axyridis using two laboratory experiments. For both no-choice and choice experiments, the probability of mating and mating duration were significantly affected by female status (alive, 1-day, 7-day, or 14-day old carcass) and by male mating status (unmated or mated) which was investigated in the no-choice experiment. The presence of chemical cues produced by an alive female did not affect the probability of a male mating with a dead female. In the no-choice experiment, 14-day old female carcasses were significantly less attractive than alive females, and unmated males tended to mate with females of all statuses with significantly higher probability than mated males. In the choice experiment, males showed a significant preference to mate with alive females when compared to 7-day old carcasses but did not distinguish between alive females and 1-day old carcasses. Mating latency (time to the starting of mating) tended to be longer for 14-day old carcasses in the no-choice experiment and was not affected by female status in the choice experiment. Mating duration was negatively affected by age of female carcasses in the no-choice experiment and only 7-day old carcasses were mated for significantly shorter time than alive females in the choice experiment. Mating behaviour was highly consistent for the no-choice and choice experiments. Despite decreasing probability of mating with 7-day and 14-day old carcasses, we observed a surprisingly high frequency of mating with dead conspecifics in the invasive ladybird Harmonia axyridis, which could have serious consequences for transmission of pathogens and affect male fitness even under natural conditions.

Age-related changes in cuticular hydrocarbon bouquet and same-sex sexual behavior of mason bee males (Osmia cornuta and O. bicornis)

Finding a mate is the central task in the lives of many male insects. In many solitary bees, sexual competition between males is high due to monandry and a pronounced male-biased sex ratio. Males searching for females will inspect and mount any object of suitable size and habitus. Species, sex, and reproductive status are encoded in the bee’s bouquet of cuticular hydrocarbons (CHCs) and can only be identified at a short distance from the object encountered. In two species of mason bees, this CHC-based system for recognizing newly emerged, virgin females is not error-free. Newly emerged individuals of both sexes carry similar CHC bouquets, facilitating the confusion of newly emerged males with newly emerged, virgin females. Misidentification leads to same-sex sexual behavior by males seeking a mate. However, individuals of both sexes modify their CHC bouquet sex-specifically within a few days. The changes in the bouquet prevent further sexual harassment. In addition, within 3 days of emergence, males begin to release species-specific carboxylic acid esters, which may act as courtship-inhibiting pheromones to extend the range of their sex-specific scent tag. Due to the pronounced protandry of both species, SSB is restricted to the early flight season, when females are not yet present, and imposes no apparent cost on the two males involved.

The endocrine disruptor DE-79 alters oxytocinergic transmission and sexual behavior expression in male rats

Polybrominated diphenyl ethers (PBDEs), used as flame retardants are persistent organic pollutants exerting important health effects. PBDEs with >5 bromide substitutions were considered less harmful and therefore extensively used commercially. DE-79 was a widely used PBDE mixture of hexa-, hepta-, octa- and nona-brominated compounds that increases vasopressin (AVP) production. AVP and oxytocin (OT) are both produced in neurons of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei projecting to the neurohypophysis and to brain regions involved in copulatory behavior. OT plays an important role in male copulation. Since DE-79 alters AVP expression in the SON and PVN, it might also modify OT content and alter male sexual behavior. We analyzed if repeated DE-79 exposure of adult male rats affected OT content and OT receptor (OTR) density in the SON, PVN, medial preoptic area (mPOA), ventral tegmental area, nucleus accumbens, and amygdala, and if male copulatory behavior was affected. We show that DE-79 exposure produces a generalized decrease in brain OT immunoreactivity, increases OTR density in all brain regions analyzed but the mPOA, and reduces the ejaculatory threshold after a first ejaculation. The documented ejaculation-induced OT release might participate in this last effect. Thus, one-week DE-79 exposure alters the OT-OTR system and modifies male rat sexual performance. Based on the literature it could be speculated that these effects are related to the putative endocrine disrupting actions of DE-79, ultimately altering brain OT levels and OTR expression that might affect copulation and other important OT-mediated brain functions.

Sequential mountings of a possible immature male by an adult male in Victoria’s Riflebird (Ptiloris victoriae)

Sexual behaviour in the Birds-of-paradise (family Paradisaeidae) is poorly understood and few studies have offered detailed descriptions of copulatory behaviours. We recorded courtship behaviours of Victoria’s Riflebird Ptiloris victoriae in the Atherton Tablelands in Queensland, Australia using motion triggered cameras and report a rare case of three sequential mountings by an adult male. While the recipient of these mountings performed female-typical sexual behaviours, it also briefly performed a male courtship display behaviour, suggesting that it may be an immature male. This observation raises several questions about the courtship behaviour of this species. Females may, for instance, occasionally solicit multiple copulations from preferred males to maximize the amount of transferred sperm. Another intriguing possibility is that immature males tolerate being mounted and potentially even mimic female sexual behaviours when learning from adult male displays. We also describe a novel adult male courtship behaviour, namely ‘nape-pecking’.

Mating and post-copulation behavior in the tea leafhopper, Empoasca onukii (Hemiptera: Cicadellidae).

The tea leafhopper, Empoasca onukii, relies on substrate-borne vibrations for sexual communication and is mainly controlled with chemical pesticides, which poses risks to the environment and food safety. Based on previous studies, we conducted a series of behavioral assays by simultaneous observation of vibration signals and movement to investigate the mating and post-copulation behavior of tea leafhoppers. During mating, the activity of E. onukii was restricted to dawn and dusk and concentrated on the sixth or seventh mature leaf below the tea bud. By comparing the time spent in locating females among different males, the timely reply of females was the key factor affecting mating success. Empoasca onukii females mated only once in their lives, while males could mate multiple times. Male rivalry behavior involved two distinct strategies. The rivals could send disruptive pulses to overlap the male calling signals, locate the courting males, and drive them away after contact. Some rivals could emit mating disruption signals (MDSs) to interrupt the ongoing identification duet and establish their own mating communication. Both identification and location duets could be interrupted by playback of MDSs, which is essential to create effective synthetic signals to disrupt mating communication of E. onukii. Our study clarified the spatial and temporal distribution of E. onukii in mating and the function of MDSs, which will be essential to develop future vibrational mating disruption techniques for E. onukii and its energy-efficient application in the field.

Male behavior in a swallowtail butterfly (Battus philenor) ensures directional iridescent sexual signal is visible to females during courtship

Male behavior in a swallowtail butterfly (Battus philenor) ensures directional iridescent sexual signal is visible to females during courtship

Sexual behavior and drive: Is it all in your brain?

Male mating behavior involves a series of behaviors aimed to recognize, approach and mate with a female. A new study in mice reveals an elaborated neural circuit that drives both sexual recognition, sexual reward, and copulatory behavior.

Genetic Diversity in Pioneer Ants: The Cardiocondyla shuckardi Group

The myrmicine ant genus Cardiocondyla consists of around 80 species of small, inconspicuous ants, which live mainly in subtropical and tropical Africa, Eurasia, and Australia. Several species have been accidentally introduced to America and have also invaded numerous originally ant-free islands around the world. The diversity of life histories in this genus, with lethally fighting wingless males, dominance hierarchies among queens, and considerable variation in the sociogenetic organization of colonies across species, has made it an interesting model to investigate the evolution and behavioral ecology of reproductive strategies. Taxa of the African Cardiocondyla shuckardi group are of particular interest, as in a phylogeny they lie between facultatively polygynous species with fatal male competition and monogynous species with mutually tolerant males. Studies on male behavior and sociogenetics in C. “venustula,” a widespread member of the C. shuckardi group, showed that males defend small territories inside their subterraneous nests. At the same time, they revealed a surprisingly large variability in mtDNA haplotypes both within and between populations in South Africa, Angola, and Côte d’Ivoire. As the different lineages are very similar in morphology and nuclear markers, more research is needed to clarify the very confused taxonomic situation in this fascinating group of ants.

Recent Trends in Cycling Behaviour in Older Males: An Analysis and Plea for Therapeutic Options

In recent years there has been an increase in middle-aged and older males riding bicycles along city streets. While this in itself would not seem to represent a problem, a number of issues arise. Firstly, these riders do not seem to venture outdoors unless they are surrounded

Two-Step Actions of Testicular Androgens in the Organization of a Male-Specific Neural Pathway from the Medial Preoptic Area to the Ventral Tegmental Area for Modulating Sexually Motivated Behavior.

The medial preoptic area (MPOA) is a sexually dimorphic region of the brain that regulates social behaviors. The sexually dimorphic nucleus (SDN) of the MPOA has been studied to understand sexual dimorphism, although the anatomy and physiology of the SDN is not fully understood. Here, we characterized SDN neurons that contribute to sexual dimorphism and investigated the mechanisms underlying the emergence of such neurons and their roles in social behaviors. A target-specific neuroanatomical study using transgenic mice expressing Cre recombinase under the control of Calb1, a gene expressed abundantly in the SDN, revealed that SDN neurons are divided into two subpopulations, GABA neurons projecting to the ventral tegmental area (VTA), where they link to the dopamine system (CalbVTA neurons), and GABA neurons that extend axons in the MPOA or project to neighboring regions (CalbnonVTA neurons). CalbVTA neurons were abundant in males, but were scarce or absent in females. There was no difference in the number of CalbnonVTA neurons between sexes. Additionally, we found that emergence of CalbVTA neurons requires two testicular androgen actions that occur first in the postnatal period and second in the peripubertal period. Chemogenetic analyses of CalbVTA neurons indicated a role in modulating sexual motivation in males. Knockdown of Calb1 in the MPOA reduced the intromission required for males to complete copulation. These findings provide strong evidence that a male-specific neural pathway from the MPOA to the VTA is organized by the two-step actions of testicular androgens for the modulation of sexually motivated behavior.SIGNIFICANCE STATEMENT The MPOA is a sexually dimorphic region of the brain that regulates social behaviors, although its sexual dimorphism is not fully understood. Here, we describe a population of MPOA neurons that contribute to the sexual dimorphism. These neurons only exist in masculinized brains, and they project their axons to the ventral tegmental area, where they link to the dopamine system. Emergence of such neurons requires two testicular androgen actions that occur first in the postnatal period and second in the peripubertal period. These MPOA neurons endow masculinized brains with a neural pathway from the MPOA to the ventral tegmental area and modulate sexually motivated behavior in males.

Reproductive payoffs of territoriality are snow‐dependent in a mountain ungulate, the Alpine chamois

AbstractFemale density and distribution are dependent on resource phenology and female availability strongly influences male mating behaviour and success. When a male adopts a ‘resource defence’ tactic, his reproductive success depends on the location and attractiveness of his territory. Environmental factors associated with territory quality are expected to influence mating success, for example, through territory features or male–male competition. In a protected population of a mountain‐dwelling polygynous herbivore, the Alpine chamois Rupicapra r. rupicapra, we investigated the relationships among mating opportunities, some environmental variables (snow depth, topographic features and size of territories) and male intra‐sexual competition for mating. We recorded the mating behaviour and territory size of 15 GPS‐GSM radio‐tagged territorial males, during five rutting seasons (early November to early December: N = 8 individuals in 2011, N = 9 in 2012, N = 8 in 2015, N = 11 in 2016, N = 7 in 2017; 80% of them were observed for more than one mating season) and related them to snow depth and topography of territories. In ruts with deep snow cover, territorial males had smaller territories and higher number of mating opportunities than in ruts with lower snow cover. Smaller territories showed the highest values of terrain roughness, in turn with little or no snow cover in the mating season, and were visited by a greater number of females, than larger territories. Number of wins was positively influenced by snow depth and negatively related to the frequency of aggressions. The frequency of male–male aggressive interactions was greater during ruts with deep snow cover and for males with territories at higher elevations; additionally, it was negatively related to interactions won. Thus, snow depth, which influences resource distribution and female movements, is confirmed as a strong determinant of male mating opportunities and mating behaviour.

Unraveling the Significance of Draglines: Female Sexual Signalization in the Nursery-Web Spider, Pisaura mirabilis.

Chemical signals used by animals to attract the opposite sex are well known in insects, but heavily understudied in spiders. We investigated the role of chemical signals in female draglines in a gift-giving spider, Pisaura mirabilis, using combined data from behavioral tests and high-performance liquid chromatography (HPLC). We also investigated whether the quality of sexual signalization is influenced by crucial factors, such as female spider ontogeny, nutritional status, and mating status. We found that draglines of adult (versus subadult) and hungry (versus fed) females stimulated male motivation to produce nuptial gift, and highly sexually excited males invested more silk in gift production than less sexually excited males. Unexpectedly, chemical signals of eggsac-carrying females were similarly sexually attractive to draglines of adult females not carrying eggsac. HPLC identified significant chemical differences in female draglines, but these differences did not always correspond to male behavior. The integration of behavioral and chemical approaches is required to better understand animal behavior in future research.

Repeated Mild Traumatic Brain Injury and JZL184 Produce Sex-Specific Increases in Anxiety-Like Behavior and Alcohol Consumption in Wistar Rats.

Alcohol use disorder (AUD) is highly comorbid with traumatic brain injury (TBI). Previously, using a lateral fluid percussion model (LFP) (an open-head injury model) to generate a single mild to moderate traumatic brain injury (TBI) we showed that TBI produces escalation in alcohol drinking, that alcohol exposure negatively impacts TBI outcomes, and that the endocannabinoid degradation inhibitor (JZL184) confers significant protection from behavioral and neuropathological outcomes in male rodents. In the present study, we used a weight drop model (a closed-head injury model) to produce repeated mild TBI (rmTBI; three TBIs separated by 24 hours) in male and female rats to examine the sex-specific effects on anxiety-like behavior and alcohol consumption, and whether systemic treatment with JZL184 would reverse TBI effects on those behaviors. In two separate studies, adult male and female Wistar rats were subjected to rmTBI or sham procedure using the weight drop model. Physiological measures of injury severity were collected from all animals. Animals in both studies were allowed to consume alcohol using an intermittent 2-bottle choice procedure (12 pre-TBI sessions and 12 post-TBI sessions). Neurological severity and neurobehavioral scores (NSS and NBS, respectively) were tested 24 hours after the final injury. Anxiety-like behavior was tested at 37-38 days post-injury in Study 1-, and 6-8-days post-injury in Study 2. Our results show that females exhibited reduced respiratory rates relative to males with no significant differences between Sham and rmTBI, no effect of rmTBI or sex on righting reflex, and increased neurological deficits in rmTBI groups in both studies. In Study 1, rmTBI increased alcohol consumption in female but not male rats. Male rats consistently exhibited higher levels of anxiety-like behavior than females. The rmTBI did not affect anxiety-like behavior 37-38 days post-injury. In Study 2, rmTBI once again increased alcohol consumption in female but not male rats, and repeated systemic treatment with JZL184 did not affect alcohol consumption. Also in Study 2, rmTBI increased anxiety-like behavior in males but not females and repeated systemic treatment with JZL184 produced an unexpected increase in anxiety-like behavior 6-8 days post-injury. In summary, rmTBI increased alcohol consumption in female rats, systemic JZL184 treatment did not alter alcohol consumption, and both rmTBI and systemic JZL184 treatment increased anxiety-like behavior 6-8 days post-injury in males but not females, highlighting robust sex differences in rmTBI effects.

Copulatory courtship, body temperature and infection in Tenebrio molitor.

Ectothermic animals can raise their body temperature under varying circumstances. Two such situations occur during sexual activity (as metabolic rate rises during copulatory movements) and during infection (to control pathogens more effectively). We have investigated these two situations using Tenebrio molitor males. We recorded the copulatory courtship behavior of sick (= infected with Metharizium robertsii fungus) vs healthy males and its link with body temperature. We predicted a positive relation between copulatory courtship (measured as antennal and leg contact behavior) and body temperature, especially in sick males. We found that the intensity of contacts correlated with increased body temperature in sick males. Previous studies in this species indicated that partner females laid fewer eggs after mating with sick males above a certain male body temperature threshold. Thus, our present findings suggest that females may detect male infection via intensity of antennal-mediated courtship, body temperature or their combination. If this is the case, females may assess male cues directly related to health status such as body temperature.

The effect of short-term exposure to high temperatures on male courtship behaviour and mating success in the fruit fly Drosophila virilis

Human-induced climate change is leading to higher average global temperatures and increasingly extreme weather events. High temperatures can have obvious effects on animal survival, particularly in ectotherms. However, the temperature at which organisms become sterile may be significantly lower than the temperature at which other biological functions are impaired. In the fruit fly Drosophila virilis, males are sterilized at temperatures above 34 °C, but are still active and able to mate normally. We investigated the male behavioural changes associated with high-temperature fertility loss. We exposed males to a warming treatment of 34.4 °C or 36.6 °C for 4 h, and then recorded their mating behaviour after being allowed to recover for 24 h. Previous work in this species suggests that males exposed to 34.4 °C lose the ability to produce new sperm, but can utilize mature sperm produced before the heat shock. We therefore predicted that these males would increases their courtship rate, and reduce their choosiness, in order to try to ensure a mating before their remaining mature sperm die. In contrast, over two-thirds of males exposed to 36.6 °C are completely sterile. In standard mating trials, earlier exposure to 34.4 °C or 36.6 °C did not affect male courtship behaviour when compared to control males kept at 23 °C. Exposure to high temperatures also did not alter the extent to which males directed courtship toward females of the same species. However, males exposed to 36.6 °C were significantly slower to mate, and had a reduced likelihood of mating, when compared to control males. Overall, exposure to high temperatures did not alter male courtship behaviour, but did lower their likelihood of mating. This suggests that females can distinguish between normal and heat-sterilized males before mating, and that female mate choice may at least partly mitigate the population-level consequences of high-temperature induced male sterility in this species.

Abstract 027: Chronic Unpredictable Stress Affects Mitochondrial Function In The Hypothalamic-Pituitary-Adrenal Axis And Impairs Vascular Reactivity In Male And Female Mice

Chronic stress is associated with hypertension but the underlying mechanisms linking the interplay between the changes in the hypothalamic-pituitary-adrenal axis and vascular dysfunction remain unclear. We hypothesize that chronic unpredictable stress (CUS) induces mitochondrial dysfunction in the HPA axis and in the vasculature, associated with GasderminD pore formation in the mitochondrial membrane. Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of CUS or handling (CTL). Anxiety-like behavior, coping behavior, and systolic blood pressure (SBP) were assessed. Mitochondrial function was assessed using high-resolution respirometry in the hypothalamus, amygdala, and adrenal tissues. The vascular reactivity of mesenteric resistance arteries (MRA), pudendal arteries (PA), and aorta were assessed in response to phenylephrine or acetylcholine. GasderminD protein expression was measured in the MRA. Data are presented as mean ± S.E.M and significance was set at p<0.05. In both sexes, CUS increased SBP by 12.1±4.9%. CUS decreased body weight gain by 53% (p=0.04) and increased anxiety-like behavior (p=0.01) in males (vs CTL) but did not affect females. CUS decreased latency to immobility in the forced swim test by 46% (p=0.0001) in both sexes. Sex-dependent decreases in mitochondrial respiration were observed as follows: in the amygdala of males (20%, p=0.05) and the hypothalamus (20%, p=0.004) and adrenal glands (17%, p=0.06) of females. In the vasculature, CUS led to endothelial dysfunction observed by reduced potency of ACh in the MRA (pEC 50 CUS 6.17±0.09 vs CTL 6.81±0.11; p=0.003) and PA (pEC 50 CUS 6.31 ±0.07 vs CTL 6.65±0.09; p=0.008) of male mice, as well as in the MRA of females (pEC 50 CUS 5.94±0.30 vs CTL 6.98±0.15; p=0.041). Contractile responses to PE were significantly reduced in the aorta of CUS males only (E MAX CTL: 4.70±0.48 mN vs CUS 3.01± 0.23 mN; p=0.007). The expression of gasderminD was increased in the MRA of female mice only. CUS increased SBP in males and females, induced sex-specific changes in the mitochondrial function in key areas of the brain, and caused vascular dysfunction, suggesting that behavioral, brain, and vascular adaptations to CUS are sex-dependent.

Social regulation of androgenic hormones and gestural display behavior in a tropical frog

Many animals use forms of gesture and dance to communicate with conspecifics in the breeding season, though the mechanisms of this behavior are rarely studied. Here, we investigate the hormone basis of such visual signal behavior in Bornean rocks frogs (Staurois parvus). Our results show that males aggregating at breeding waterfalls have higher testosterone (T) levels, and we speculate that this hormone increase is caused by social cues associated with sexual competition. To this end, we find that T levels in frogs at the waterfall positively predict the number waving gestures—or “foot flags”—that males perform while competing with rivals. By contrast, T does not predict differences in male calling behavior. In these frogs, vocal displays are used largely as an alert signal to direct a rival's attention to the foot flag; thus, our results are consistent with the view that factors related to reproductive context drive up T levels to mediate displays most closely linked to male-male combat, which in this case is the frog's elaborate gestural routine.