Urban areas are characterised by the presence of sensory pollutants, such as anthropogenic noise and artificial light at night (ALAN). Animals can quickly adapt to novel environmental conditions by adjusting their behaviour, which is proximately regulated by endocrine systems. While endocrine responses to sensory pollution have been widely reported, this has not often been linked to changes in behaviour, hampering the understanding of adaptiveness of endocrine responses. Our aim was, therefore, to investigate the effects of urbanisation, specifically urban noise and light pollution, on hormone levels in male urban and forest túngara frogs (Engystomops pustulosus), a species with reported population divergence in behaviour in response to urbanisation. We quantified testosterone and corticosterone release rates in the field and in the lab before and after exposure to urban noise and/or light. We show that urban and forest frogs differ in their endocrine phenotypes under field as well as lab conditions. Moreover, in urban frogs exposure to urban noise and light led, respectively, to an increase in testosterone and decrease in corticosterone, whereas in forest frogs sensory pollutants did not elicit any endocrine response. Our results show that urbanisation, specifically noise and light pollution, can modulate hormone levels in urban and forest populations differentially. The observed endocrine responses are consistent with the observed behavioural changes in urban frogs, providing a proximate explanation for the presumably adaptive behavioural changes in response to urbanisation.

Gonadal steroid hormones are typically elevated during the breeding season—a finding known as an associated reproductive pattern. Though less studied, there is also evidence, in both sexes, for elevated adrenal/interrenal steroids, including acute elevations on the day of mating. I investigated gonadal and interrenal steroids in wild male Cope’s gray treefrogs at breeding aggregations. I collected blood from males found in amplexus with female mates (amplexed males) and males sampled at the same time and location that were actively advertising vocally and without a mate (solo males). Concentrations of plasma corticosterone, testosterone, and 17b-estradiol (CORT, T and E₂, respectively) were validated and measured. These two categories of males differed in four ways: (1) amplexed males exhibited significantly elevated concentrations of all three steroids compared to solo males (CORT: +347%; T: +60%; and E₂: +43%); (2) these hormone profiles alone accurately predicted male mating category with ca. 83% accuracy using a discriminant function analysis; (3) amplexed males exhibited significant between-hormone correlations (T and E₂ were positively correlated and CORT and E₂ were negatively correlated) whereas no correlations were found in solo males; (4) amplexed males showed a negative correlation with CORT concentration and the time of night, whereas no such pattern was present in solo males. These findings suggest an acute and strong coactivation of the interrenal and gonadal axes that could drive phenotypic integration during this fitness-determining moment. I discuss these findings and suggest experiments to determine causation, including the role of motor behavior driving endocrine states and the role of female selection on endocrine profiles.

Selecting an attractive mate can involve trade-offs related to investment in sampling effort. Glucocorticoids like corticosterone (CORT) are involved in resolving energetic trade-offs. However, CORT is rarely studied in the context of mate choice, despite its elevated levels during reproductive readiness and the energetic transitions that characterize reproduction. Few systems are as well suited as anuran amphibians to evaluate how females resolve energetic trade-offs during mate choice. Phonotaxis tests provide a robust bioassay of mate choice that permit the precise measurement of inter-individual variation in traits such as choosiness–the willingness to pursue the most attractive mate despite costs. In Cope’s gray treefrogs (Hyla chrysoscelis), females exhibit remarkable variation in circulating CORT as well as choosiness during mate choice, and a moderate dose of exogenous CORT rapidly (<1 h) and reliably induce large increases in choosiness. Here we measured the expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors in the brains of females previously treated with exogenous CORT and tested for mate choosiness. We report a large decrease in GR expression in the hindbrain and midbrain of females that were treated with the moderate dosage of CORT–the same treatment group that exhibited a dramatic increase in choosiness following CORT treatment. This association, however, does not appear to be causal, as only forebrain GR levels, which are not affected by CORT injection, are positively associated with variation in choosiness. No strong effects were found for MR. We discuss these findings and suggest future studies to test the influence of glucocorticoids on mate choice.

Abstract  Most animals experience reproductive transitions in their lives; for instance, reaching reproductive maturity or cycling in and out of breeding condition. Some reproductive transitions are abrupt, while others are more gradual. In most cases, changes in communication between the sexes follow the time course of these reproductive transitions and are typically thought to be coordinated by steroid hormones. We know a great deal about hormonal control of communication behaviors in birds and frogs, as well as the central neural control of these behaviors. There has also been significant interest in the effects of steroid hormones on central nervous system structures that control both the production and reception of communication signals associated with reproductive behaviors. However, peripheral sensory structures have typically received less attention, although there has been growing interest in recent years. It is becoming clear that peripheral sensory systems play an important role in reproductive communication, are plastic across reproductive conditions, and, in some cases, this plasticity may be mediated by steroid hormones. In this paper, we discuss recent evidence for the role of peripheral auditory structures in reproductive communication in birds and frogs, the plasticity of the peripheral auditory system, and the role of steroid hormones in mediating the effects of the peripheral auditory system on reproductive communication. We focus on both seasonal and acute reproductive transitions, introduce new data on the role of hormones in modulating seasonal patterns, and make recommendations for future work.

Abstract. Glucocorticoids (GCs) are rarely studied in the context of female mate choice, despite the expression of receptors for these products in sexual, sensory and decision-making brain areas. Here we investigated the effects of GC concentrations on three aspects of female sexual behavior in breeding Cope’s gray treefrogs (Hyla chrysoscelis): proceptivity—a measure of sexual motivation, intraspecific mate preferences, and mate choosiness. To our knowledge this is the first experimental study on the endocrine basis of mate choosiness. We predicted that mate choosiness—forfeiting an initial mate preference to pursue a suddenly more attractive mate—would be particularly impacted by elevated GCs with moderate GC levels associated with greater choosiness. We found support for this predicted inverted-U relationship. Females in the control group (no injection) showed no change in choosiness across timepoints. In contrast, females in the vehicle, Low (20 ng g^-1) and High (180 ng g^-1) corticosterone groups exhibited a nominal decline in choosiness after injection, suggesting that the experience of injection has little or perhaps slightly suppressive effects on female choosiness. Females in the moderate dose group (60 ng g^-1), however, exhibited a significant increase (>100%) in choosiness. Further, we found no effect of elevated GCs on sexual proceptivity or the species-typical preference for longer calls. These findings may reflect a buffering of primary sensory areas in the brain against elevated GCs. The recruitment of other cognitive processes during active decision-making, however, may facilitate GC modulation of mate choosiness, thereby promoting tactical plasticity at this critical life history juncture.

Abstract  Conventional methods for sampling hormones often preclude strong inference experimental designs, including repeated measures of both hormones and behavior and balanced or simultaneous designs for hormone-behavior sampling. In amphibians there is an opportunity to non-invasively and repeatedly sample excreted steroids in the water. We examined testosterone (T) in túngara frogs (Physalaemus (=Engystomops) pustulosus) using minimally invasive water-borne methods. First, we validated procedures for the collection, extraction and measurement of T in adult males and females using pharmacological challenges coupled with estimates of parallelism and recovery determination. Next, we extended the timeline of sampling over 9 days in order to evaluate the kinetics of excretion (baseline phase, challenge phase, recovery phase), including the estimation of individual differences during baseline sampling. We also estimated concentrations of creatinine (Cr) in each water sample and evaluated whether correcting for this proxy of urine concentration significantly decreased error variance in T estimates. Lastly, we incorporated a standardized and repeated measures assay of male sexual proceptivity (phonotaxis) during the predicted peak T and recovery T timepoints. We found strong evidence supporting the utility of these methods for precise, biologically informative estimates of T in both sexes. Males had higher T than females and responded to pharmacological challenges by elevating T substantially within 48 h of challenge (hCG, GnRH). Males exhibited repeatability in baseline T and phonotaxis frequencies were positively associated with higher T. Adjusting T levels for the simultaneous measure of Cr significantly improved model fit, which in conjunction with marked variation in urine concentration, suggests that urine likely serves as the major source of excreted T. In summary, this proof-of-concept and methods study demonstrates the utility and accuracy of measuring water-borne T and behavior in amphibians.

Abstract  We review work relating glucocorticoids (GCs), male sexual signals, and mate choice by females to
understand the potential for GCs to modulate the expression of sexually selected traits and how sexual
selection potentially feeds back on GC regulation. Our review reveals that the relationship between GC
concentrations and the quality of male sexual traits is mixed, regardless of whether studies focused on
structural traits (e.g., coloration) or behavioral traits (e.g., vocalizations) or were examined in
developmental or activational frameworks. In contrast, the few mate choice experiments that have been
done consistently show that females prefer males with low GCs, suggesting that mate choice by females
favors males that maintain low levels of GCs. We point out, however, that just as sexual selection can
drive the evolution of diverse reproductive strategies, it may also promote diversity in GC regulation. We
then shift the focus to females where we highlight evidence indicating that stressors or high GCs can
dampen female sexual proceptivity and the strength of preferences for male courtship signals. Hence,
even in cases where GCs are tightly coupled with male sexual signals, the strength of sexual selection on
aspects of GC physiology can vary depending on the endocrine status of females. Studies examining how
GCs relate to sexual selection may shed light on how variation in stress physiology, sexual signals, and
mate choice are maintained in natural populations and may be important in understanding context-
dependent relationships between GC regulation and fitness.

Abstract  Both behavioral receptivity and neural sensitivity to acoustic mate attraction signals vary across the reproductive cycle, particularly in seasonally breeding animals. Across a variety of taxa receptivity to signals increases, as does peripheral auditory sensitivity, as females transition from a non-breeding to breeding condition. We recently documented decreases in receptivity to acoustic mate attraction signals and circulating hormone levels, but an increase in peripheral auditory sensitivity to call-like stimuli following oviposition in Cope’s gray treefrogs (Hyla chrysoscelis). However, it is not known if changes in auditory sensitivity are confined to the frequency range of calls, or if they result from more generalized changes in the auditory periphery. Here, we used auditory brainstem responses (ABRs) to evaluate peripheral frequency sensitivity in female Cope’s gray treefrogs before and after oviposition. We found lower ABR thresholds, greater ABR amplitudes, and shorter ABR latencies following oviposition. Changes were most pronounced and consistent at lower frequencies associated with the amphibian papilla, but were also detectable at higher frequencies corresponding to the tuning of the basilar papilla. Furthermore, only ABR latencies were correlated with circulating steroid hormones (testosterone). Changes in peripheral processing may result from changes in metabolic function or sensorineural adaptation to chorus noise.

Abstract  In seasonal breeders, there are behavioral, endocrine, and neural adaptations that promote the sexual receptivity of females and tune their sensory systems to detect and discriminate among advertising males and to successfully copulate. What happens immediately after this key life history event is unclear, but this transitional moment offers a window into the mechanisms that remodel sexual phenotypes. In this study of wild female Cope’s gray treefrogs (Hyla chrysoscelis), we tested the hypothesis that oviposition results in a suite of coordinated changes in the sexual phenotype. Specifically, we predicted that sexual receptivity and discrimination behaviors would decline along with circulating concentrations of steroid hormones (corticosterone, estradiol, testosterone) and auditory sensitivity to the acoustic frequencies emphasized in male advertisement calls. We conducted these trait measurements before and after oviposition (ca. 24-hr period). There was a 100% decrease in behavioral responsiveness after oviposition, and the concentrations of all three steroids plummeted during this brief window of time, especially testosterone. Moreover, higher concentrations of corticosterone—an important component of the endocrine stress response—were associated with longer response latencies, suggesting that adrenal hormones should be considered in future studies on the hormonal basis of mate choice. Counter to our prediction, auditory sensitivity increased following oviposition, and the amplitude of the auditory brainstem response was influenced by concentrations of estradiol. In pre-oviposition females auditory sensitivity diminished with increasing estradiol concentrations, while sensitivity increased with increasing estradiol concentrations in post-oviposition females, suggesting non-linear estrogenic modulation of peripheral auditory neural recruitment. Overall, our results indicate that there is considerable remodeling of behavioral output following oviposition that co-occurs with changes in both endocrine and sensory physiology.

Abstract  Sexual selection driven by mate choice has generated some of the most astounding diversity in nature, suggesting population-level preferences should be strong and consistent over many generations. On the other hand, mating preferences are among the least repeatable components of an individual animal’s phenotype, suggesting low consistency across an animal’s lifetime. Despite decades of intensive study of sexual selection there is almost no information about the strength and consistency of preferences across many years. In this study we present the results of over 5000 mate choice tests with a species of wild frog conducted over 19 consecutive years. Results show that preferences are positive and strong and vary little across years. This consistency is despite the fact that there are substantial differences among females in their strength of preference. We also suggest mate preferences that are primarily the result of sensory exploitation might be more stable in populations compared to preferences that are primarily involved in assessing male quality.

Abstract  Minimally invasive methods for estimating hormone concentrations in wild vertebrates offer the opportunity to repeatedly measure behavior and hormone concentrations within individuals while minimizing experimenter interference during sample collection. We examined three steroid hormones (corticosterone, CORT; 17-b estradiol, E₂; progesterone, PROG) in túngara frogs (Physalaemus pustulosus) using non-invasive water-borne methods. Using solid-phase extraction of water samples and liquid extraction of plasma and homogenate samples, coupled with enzyme immunoassays, we complimented the conventional validation approaches (parallelism, recovery determination) with dose-response assays that incorporated pharmacological challenges with adrenocorticotropic hormone (ACTH) and human chorionic gonadotropin (HCG). We also compared steroid concentrations in water to those observed in plasma and whole body homogenates. Lastly, we identified the constituent steroids in each sample type with a panel targeting 30 steroid species using high performance liquid chromatography-mass spectrometry (HPLC-MS). We found that a 60-min water-bath captures physiologically relevant changes in concentrations of CORT, E₂ and PROG. Peak levels of water-borne CORT were found at approximately 2 h after ACTH injection. Water-borne CORT and E₂ concentrations were positively correlated with their plasma and homogenate equivalents, while water-borne PROG was uncorrelated with homogenate PROG concentrations but negatively correlated with homogenate E₂ concentrations. Together, our findings indicate that sampling water-borne hormones presents a non-invasive and biologically informative approach that will be useful for behavioral endocrinologists and conservation physiologists.

Abstract  Strong sexual selection by receivers can lead to the evolution of elaborate courtship behaviors in signalers. However, the process by which receivers sample signalers and execute mate choice under complex signaling conditions—and thus the realized strength of sexual selection—is poorly understood. Moreover, receivers can vary in condition, which can further influence mate sampling strategies. Using wild female frogs we tested two hypotheses at the intersection of these important problems: that some of the individual variation in mate sampling is explained by (1) the reproductive urgency hypothesis, which predicts that receivers in a more urgent reproductive state will sample mates less and/or (2) the reproductive investment hypothesis, which predicts that receivers that have invested less in the current reproductive effort will sample mates less. Eastern gray treefrogs, Hyla versicolor, were collected in amplexus and repeatedly tested for phonotaxis behavior using a dynamic playback assay. To evaluate if hormonal mechanisms explained variation in the mate sampling, three steroid hormones, estradiol, progesterone, and corticosterone, were collected using a noninvasive water-borne hormone assay, validated for this species in the present study. Finally, we measured clutch size (investment) and the duration of time required for each female to oviposit after being reunited with their male mate (urgency). We found repeatability in many of the behaviors, including mate sampling. We found that females with higher concentrations estradiol and corticosterone made quicker choices, and that females with higher progesterone sampled mates more. We also found that female frogs in a more urgent reproductive state had lower concentrations of progesterone and estradiol, thereby providing the first evidence of a relationship between gonadal hormones and reproductive urgency. Collectively we found some support for the reproductive urgency but not the investment hypothesis. Thus, even though a female frog’s reproductive readiness is a highly transient life history stage, fine scale variation in her reproductive timeline could mitigate the strength of directional selection.

Abstract  The evolution of elaborate signals can emerge from changes in anatomical signaling structures. In the male túngara frog, a simple advertisement call (‘whine’) can be ornamented facultatively with a suffix (‘chuck’) to produce a more attractive complex call, or ‘whinechuck’. A fibrous mass (FM1) supported by the vocal cords plays a role in chuck production. Here, we examine the effects of FM1 ablation on a large set of spectral and temporal features of both the whine and chuck and we test the hypothesis that FM1 ablation reduces call attractiveness to females. Both call components were impacted by FM1 ablation, but especially the suffix. The proportion of energy in the suffix’s odd compared to even harmonics diminished markedly as did the relative amplitude, effectively eliminating the chuck percept. FM1 ablation also reduced the whine’s frequency and its rate of frequency modulation. Moreover, post-surgery chucks no longer enhanced the attractiveness of the simple call and whines also appeared to diminish in attractiveness. Together, our results demonstrate that FM1 plays a role in the production of both call components in a way that stimulates the female auditory system and that the growth of FM1 had potentially positive sexual selection implications for the preexisting simple call.

Abstract   The neuropeptide arginine vasotocin (AVT) promotes sexual advertisement and influences vocalization structure in male anuran amphibians. In the present study, we used wild túngara frogs (Physalaemus pustulosus) to investigate the effects of AVT on phonotaxis in males and females—thereby controlling for potential task differences between the sexes. Using a combined within- and between-subjects design, we showed that acoustic choice behavior in female frogs is not influenced by injection per se (vehicle) or by AVT. Latency to choice in females, however, tends to decrease after AVT injection, supporting the hypothesis that AVT promotes female sexual arousal. In contrast, male choice behavior and latencies are negatively impacted by injection (vehicle) but rescued to pre-injection levels if administered with AVT. The sexes differed in area restricted searching (ARS) following choice—a measure of locomotor perseverance—with females but not males exhibiting ARS. AVT did not influence ARS behavior but ARS frequency was positively associated with the attractiveness of the acoustic stimulus. Finally, we showed that a female’s latency behavior is correlated with her partner’s behavior. Collectively we show that AVT promotes phonotaxis in both sexes in a dimorphic manner—a result that is consistent with sex differences in the neural vasotocin system.

Abstract   Variation in the reactivity of the endocrine stress axis is thought to underlie aspects of persistent individual differences in behavior (i.e. animal personality). Previous studies, however, have focused largely on estimating baseline or peak levels of glucocorticoids (CORT), often in captive animals. In contrast, the temporal dynamics of the HPA axis—how quickly it turns on and off, for example—may better indicate how an individual copes with stressors. Moreover, these HPA components might be correlated, thereby representing endocrine suites. Using wild-caught great tits (Parus major) we tested birds for exploratory behavior using a standardized novel environment assay that serves as a validated proxy for personality. We then re-captured a subset of these birds (n = 85) and characterized four components of HPA physiology: baseline, endogenous stress response, a dexamethasone (DEX) challenge to estimate the strength of negative feedback, and an adrenocorticotropic hormone (ACTH) challenge to estimate adrenal capacity. We predicted that these four HPA responses would be positively correlated and that less exploratory birds would have a more rapid onset of the stress response (a CORT elevation during the baseline bleed) and weaker negative feedback (higher CORT after DEX). We found support for the first two predictions but not the third. All four components were positively correlated with each other and less exploratory birds exhibited an elevation in CORT during the baseline bleed (<3 min from capture). Less exploratory birds, however, did not exhibit weaker negative feedback following the DEX challenge, but did exhibit weaker adrenal capacity. Together, our findings provide partial support for the hypothesis that the temporal reactivity of the HPA axis is linked with consistent individual differences in behavior, with more cautious (slower exploring) individuals exhibiting a faster CORT response.

Abstract  Hormonal pleiotropy—the simultaneous influence of a single hormone on multiple traits—has been hypothesized as an important mechanism underlying personality, and circulating glucocorticoids are central to this idea. A major gap in our understanding is the neural basis for this link. Here we examine the stability and structure of behavioral, endocrine and neuroendocrine traits in a population of songbirds (Parus major). Upon identifying stable and covarying behavioral and endocrine traits, we test the hypothesis that risk-averse personalities exhibit a neuroendocrine stress axis that is systemically potentiated—characterized by stronger glucocorticoid reactivity and weaker negative feedback. We show high among-individual variation and covariation (i.e. personality) in risk-taking behaviors and demonstrate that four aspects of glucocorticoid physiology (baseline, stress response, negative feedback strength and adrenal sensitivity) are also repeatable and covary. Further, we establish that high expression of mineralocorticoid and low expression of glucocorticoid receptor in the brain are linked with systemically elevated plasma glucocorticoid levels and more risk-averse personalities. Our findings support the hypothesis that steroid hormones can exert pleiotropic effects that organize behavioral phenotypes and provide novel evidence that neuroendocrine factors robustly explain a large fraction of endocrine and personality variation.

Abstract   Stress physiology is thought to contribute to individual differences in behaviour. In part this reflects the fact that canonical personality measures consist of responses to challenges, including novel objects and environments. Exposure to novelty is typically assumed to induce a moderate increase in glucocorticoids (CORT), although this has rarely been tested. We tested this assumption using great tits, Parus major, selected for divergent personalities (bold-fast and shy-slow explorers), predicting that the shy birds would exhibit higher CORT following exposure to a novel object. We also scored behavioural responses to the novel object, predicting that bold birds would more frequently approach the novel object and exhibit more abnormal repetitive behaviours. We found that the presence of a novel object did induce a moderate CORT response, but selection lines did not differ in the magnitude of this response. Furthermore, although both selection lines showed a robust CORT elevation to a subsequent restraint stressor, the CORT response was stronger in bold birds and this effect was specific to novel object exposure. Shy birds showed a strong positive phenotypic correlation between CORT concentrations following the novel object exposure and the subsequent restraint stress. Behaviourally, the selection lines differed in their response during novel object exposure: as predicted, bold birds more frequently approached the novel object and shy birds more strongly decreased overall locomotion during the novel object trial, but birds from both selection lines showed significant and similar frequencies of abnormal repetitive behaviours during novel object exposure. Our findings support the hypothesis that personality emerges as a result of correlated selection on behaviour and underlying endocrine mechanisms and suggest that the relationship between endocrine stress physiology and personality is context dependent.

Abstract 

1. Animal personality traits emerge developmentally from the interaction of genetic and

early environmental factors. Maternal hormones, such as androgens (testosterone, T and

androstenedione, A4), transferred to embryos and egg yolks may simultaneously organize

multiple behavioural and physiological traits. Whereas previous studies demonstrated an

association between the mother’s personality and yolk androgen levels, the independent

effects of the male partner’s personality and pair combination are unknown.

2. We test this association using an ecological model species for personality research, the

great tit (Parus major) using multiple approaches: 1) a wild population, 2) a randomly

mated captive population, and 3) an experimental study with (dis)assortatively mated

pairs from lines selected for fast exploration/boldness or slow exploration/shyness.

3. Egg androgen concentrations were associated with variation in female personality traits,

and the experimental data suggested that this is independent of male personality:

experimental females from slow-shy line tended to have higher egg T concentrations than

females from the fast-bold line, with no effect of male personality. Shy females from the

wild population had higher egg A4 concentration than bold females. However, in the

correlative data yolk hormones were linked with male personality, as well as the

interaction between female and male traits and yolk androgen levels: Male handling

responsiveness correlated negatively with egg A4 concentration in wild birds. In

randomly-mated birds, pairs that were mated assortatively for personality had lower egg

T concentrations than disassortatively mated pairs.

4. Given that egg androgens are known developmental mediators of avian personality, our

results suggest that maternal hormones might contribute to the heritability of personality,

may be sensitive to the social context of mating, and act as key environmental drivers of

individual differences.

Abstract  In humans and some nonhuman vertebrates, a sound containing brief silent gaps can be rendered perceptually continuous by inserting noise into the gaps. This so-called “continuity illusion” arises from a phenomenon known as “auditory induction” and results in the perception of complete auditory objects despite fragmentary or incomplete acoustic information. Previous studies of auditory induction in gray treefrogs (Hyla versicolor and H. chrysoscelis) have demonstrated an absence of this phenomenon. These treefrog species produce pulsatile (noncontinuous) vocalizations, whereas studies of auditory induction in other taxa, including humans, often present continuous sounds (e.g., frequency-modulated sweeps). This study investigated the continuity illusion in a frog (Physalaemus pustulosus) with an advertisement vocalization that is naturally continuous and thus similar to the tonal sweeps used in human psychophysical studies of auditory induction. In a series of playback experiments, female subjects were presented with sets of stimuli that included complete calls, calls with silent gaps, and calls with silent gaps filled with noise. The results failed to provide evidence of auditory induction. Current evidence, therefore, suggests that mammals and birds experience auditory induction, but frogs may not. This emerging pattern of taxonomic differences is considered in light of potential methodological, neurophysiological, and functional explanations.

Abstract  Endocrine systems play critical roles in facilitating sexual behavior in seasonally breeding vertebrates. Much of the research exploring this topic has focused on the endocrine correlates of signaling behavior in males and sexual proceptivity in females. What is less understood is how hormones promote the expression of the often complex and highly selective set of stimulus–response behaviors that are observed in naturally breeding animals. In female frogs, phonotaxis is a robust and sensitive bioassay of mate choice and is exhibited by gravid females during the breeding season. In stark contrast, females exhibit low phonotactic responsiveness outside the breeding season, but the administration of hormones can induce sexual proceptivity. Here we test the hypothesis that manipulation of a minimal set of reproductive hormones—progesterone and prostaglandin F2α—are capable of evoking not only proceptive behavior in non-breeding females, but also the patterns of intraspecific selectivity for male sexual displays observed in gravid females tested during the breeding season. Specifically, we investigated whether preferences for faster call rates, longer call durations, and higher call efforts were similar between breeding and hormone-treated females of Cope’s gray treefrog (Hyla chrysoscelis). Hormone injections induced patterns of selective phonotaxis in non-breeding females that were remarkably similar to those observed in breeding females. These results suggest that there may be an important contribution of hormonal pleiotropy in regulating this complex, acoustically-guided sexual behavior. Our findings also support the idea that hormonal induction could be used to evaluate hypotheses about selective mate choice, and its underlying mechanisms, using non-breeding females.

Abstract  In evolutionary endocrinology, there is a growing interest in the extent and basis of individual variation in endocrine traits, especially circulating concentrations of hormones. This is important because if targeted by selection, such individual differences present the opportunity for an evolutionary response to selection. It is therefore necessary to examine whether hormone traits are repeatable in natural populations. However, research in this area is complicated by the fact that different hormone traits can be correlated. The nature of these trait correlations (i.e., phenotypic, within-, or among-individual) is critically relevant in terms of the evolutionary implications, and these in turn, depend on the repeatability of each hormone trait. By decomposing phenotypic correlations between hormone traits into their within- and among-individual components it is possible to describe the multivariate nature of endocrine traits and generate inferences about their evolution. In the present study, we repeatedly captured individual great tits (Parus major) from a wild population and measured plasma concentrations of corticosterone. Using a mixed-modeling approach, we estimated repeatabilities in both initial (cf. baseline; CORT0) and stress-induced concentrations (CORT30) and the correlations between those traits among- and within-individuals. We found a lack of repeatability in both CORT0 and CORT30. Moreover, we found a strong phenotypic correlation between CORT0 and CORT30, and due to the lack of repeatability for both traits, there was no among-individual correlation between these two traits—i.e., an individual’s average concentration of CORT0 was not correlated with its average concentration of CORT30. Instead, the phenotypic correlation was the result of a strong within-individual correlation, which implies that an underlying environmental factor co-modulates changes in initial and stress-induced concentrations within the same individual over time. These results demonstrate that (i) a phenotypic correlation between two hormone traits does not imply that the traits are correlated among individuals; (ii) the importance of repeated sampling to partition within- and among-individual variances and correlations among labile physiological traits; and (iii) that environmental factors explain a considerable fraction of the variation and co-variation in hormone concentrations.

Abstract  Phenotypic correlations, such as those between functionally distinct behavioral traits, can emerge through the action of selection on individual traits, on trait combinations, and through pleiotropic mechanisms. Steroid hormones are known to have pleiotropic effects on a suite of behavioral and physiological traits, including stable individual differences in coping with stress. Characterizing the stress axis in relation to personality, however, has typically focused on estimating baseline and peak levels of glucocorticoids, principally in captive animals. In contrast, the reactivity of the stress response—how quickly it turns on and persists—may better indicate the ability of an individual to cope with challenges, particularly in free-living animals. Using wild great tits (Parus major) we tested the hypothesis that cautious individuals respond to a standardized stressor with a more reactive stress response compared to bolder individuals. Wild birds were captured and tested for exploration behavior in a novel environment—an operational measure of personality in this species—and assessed separately for their glucocorticoid response to a standardized stressor. Slower explorers exhibited a greater elevation in glucocorticoid levels within the first three minutes after capture. Further, slower explorers reached a higher maximum CORT concentration and had higher total exposure to glucocorticoids during the stressor period. These data provide evidence that the temporal reactivity of the endocrine stress response, specifically its speed and magnitude, is associated with stable behavioral traits in free-living animals.

Abstract  Most studies addressing the development of animal communication have focused on signal production rather than receiver decoding, and similar emphasis has been given to learning over nonlearning. But receivers are an integral part of a communication network, and nonlearned mechanisms appear to be more ubiquitous than learned ones in the communication systems of most animals. Here we review the results of recent experiments and outline future directions for integrative studies on the development of a primarily nonlearned behaviour—recognition of communication signals during ontogeny in a tropical frog. The results suggest that antecedents to adult behaviours might be a common feature of developing organisms. Given the essential role that acoustic communication serves in reproduction for many organisms and that receivers can exert strong influence on the evolution of signals, understanding the evolutionary developmental basis of mate recognition will provide new insights into the evolution of communication systems.

Abstract  Animal ‘personality’ describes consistent individual differences in suites of behaviors, a phenomenon exhibited in diverse animal taxa and shown to be under natural and sexual selection. It has been suggested that variation in personality reflects underlying physiological variation; however there is limited empirical evidence to test this hypothesis in wild animals. The hypothalamic–pituitary–adrenal axis is hypothesized to play a central role in personality variation. Here we tested whether in great tits Parus major variation in personality traits is related to plasma concentrations of corticosterone (CORT). Using a capture-restraint protocol we examined baseline and stress-induced CORT levels in two captive experimental groups: (1) birds selected for divergent personalities (‘fast-bold’ and ‘slow-shy’ explorers); and (2) non-selected offspring of wild parents. We first tested for differences in CORT between selection lines, and second examined the relationship between responses in a canonical personality test and CORT concentrations in non-selected birds. We found support for our prediction that the slow-shy line would exhibit a higher acute stress response than the fast-bold line, indicating a genetic correlation between exploratory behavior and stress physiology. We did not, however, find that continuous variation in exploratory behavior co-varies with CORT concentrations in non-selected birds. While our results provide support for the idea that personality emerges as a result of correlated selection on behavior and underlying physiological mechanisms, they also indicate that this link may be particularly evident when composite personality traits are the target of selection.

Abstract  In many species males vocally advertise for mates in choruses and these choruses serve as acoustic beacons to conspecific females as well as to eavesdropping predators and parasites. Chorusing will often cease in response to disturbances, such as the presence of predators. In some cases the cessation is so rapid and over such a large area that it seems improbable that males are all responding directly to the same local disturbance. Here, we demonstrate experimentally in Neotropical túngara frogs, Physalaemus pustulosus, that the cessation of calling by males spreads rapidly through the chorus. The cessation of chorusing in response to the cessation of playbacks of three calling males is more effective in inducing chorus cessation than is the cessation of one male calling. When three males are calling, the cessation of complex calls is more effective in inducing chorus cessation than simple calls. There is no main effect on whether the final call of the male is complete or is interrupted. We thus conclude that the sudden lack of signals—the ‘sounds of silence’—becomes an alarm cue that can explain the rapid cessation of choruses that are common in many chorusing species.

Abstract  Facultative traits that have evolved under sexual selection, such as the acoustic ornaments present in the advertisement signals of male túngara frogs (Physalaemus pustulosus), offer a unique opportunity to examine selection for trait exaggeration with a focus on individual differences amongst signalers. By contrast, many studies of mate choice use experimental designs that obscure the interindividual variation amongst signalers available for selection to act on—through the use of “typical” or average signals from the population. Here, we use dichotomous female phonotaxis choice tests to determine how the value of male call embellishment varies across 20 individual males frogs recorded from the wild—a sample which captures the acoustic diversity present in the population. We tested 20 females for each male call pair (i.e., 400 females). The results show widespread preference amongst females for ornamented calls (“whine–chucks”) over simple calls (“whines”), yet also demonstrate substantial variation in the relative benefits for individual male frogs—some males enjoy appreciable benefits by using ornaments while others (30% of males in this study) do not. We also show that the relative amplitude of the chuck to the whine correlates positively with the value of call elaborations across these 20 males. Finally, by manipulating the relative amplitude of whines and chucks using both natural and synthetic calls, we demonstrate directly that this single call parameter is key to determining the relative value of call elaborations across males.

Abstract  We examined the emergence of a critical component of sex, response to sexual signals— phonotaxis—in male and female tungara frogs (Physalaemus pustulosus). We determined the ontogenetic trajectories of phonotactic responses as animals developed from metamorphic froglets to reproductive adults. The results demonstrated that species-typical phonotaxis emerges quite early during postmetamorphic development, well before sexual maturity, suggesting that a developmentally early bias in the auditory system for species-typical signals might be a more general phenomenon than previously thought, and that the neural circuits responsible for processing and responding to conspecific advertisement signals in a species-typical manner might develop long before the coordinated behavior is demanded of the organism.

Abstract  In acoustically advertising anurans the male courtship call elicits species-typical responses from conspecifics – usually phonotactic approach and mate choice in gravid females and an evoked vocal response in adult males. Males in several species, however, are also known to perform phonotaxis, sometimes with the same acoustic preferences as females. Female túngara frogs are known to update their phonotactic approach as male advertisement signals change dynamically in attractiveness. Here we show that males also perform such temporal updating during phonotaxis in response to dynamic playbacks. While males exhibit slower phonotactic approaches than females, their responsiveness to dynamic changes in call complexity does not differ significantly compared to females. These results demonstrate that males are sensitive to the location of preferred call types on a moment-to-moment basis and suggest that similarities between male and female sexual behaviour in anurans might often be overlooked. We suggest that anuran phonotaxis is more widespread and serves different functions in reproductive females and males. Lastly, these temporal updating results suggest that male frogs are highly selective about site selection in a chorus.

Abstract  Signalling is a dynamic process that often occurs over brief timescales, particularly in the acoustic modality. Numerous studies of mate choice and acoustic communication have identified signal parameters essential for species recognition and mate preferences, although these studies have rarely considered the dynamic nature of these processes. Here we investigate mate choice behaviour in female túngara frogs, Physalaemus pustulosus, in response to temporally dynamic presentations of male advertisement calls. Our results demonstrate that females are sensitive to the location of preferred call types on a moment-to-moment basis, and that responses are influenced by the continued presence, complexity, sound pressure level and inherent attractiveness of individual male signals. In general, our results support the notion that decision making during mate choice is an open-ended process that is sensitive to interruption and persuasion from competing signallers. We show that for a species in which females actively compare multiple signallers simultaneously, the criterion that predicts the degree of commitment to an initial mate approach is whether there is a state change in the complexity of signals.

Abstract  It is well known that animal decision-making can be influenced by environmental variables, such as the risk of predation. During the breeding season, nocturnal amphibians encounter a range of environmental conditions at breeding aggregations, including variable ambient light conditions. For nocturnal frogs, illumination is expected to minimize conspicuous movement that might increase predator detection. Previous work has shown that female Physalaemus pustulosus (Cope, 1864) (= Engystomops pustulosus (Cope, 1864)) are sensitive to variation in light levels during mate choice. Here we use an acoustic playback design in which stimuli are adjusted for intensity and complexity during female phonotaxis to show that choosiness is influenced by light level. Frogs were more likely to commit to an initial mate choice despite a dynamic reduction in mate attractiveness under dim light conditions compared with darkness. These results suggest that females are trading off the attractiveness of potential mates with the perceived costs of executing mate choice by committing to an initial decision and thereby reducing assessment time and movement. The dynamic playback design used here provides an approach that could be applied in other systems in which context-dependent decision-making is thought to be important.

Abstract  Interest in the question of when and how species recognition and mate preferences emerge in animals with strong species-typical predispositions has faded since the time of the classical ethologists. In its place, the role of plasticity has surfaced as a central emphasis in the study of animal behavior. Here, I step back and examine the origin and execution of sexual behavior in a tropical frog for which auditory predispositions are key. These experiments challenge assumptions about behavioral development, auditory perception, and stereotyped behavior. First, I illustrate when and how a sex- and speciestypical behavior—conspecific phonotaxis—emerges during development. This study demonstrates that phonotaxis, presumably restricted to mature females, is present in both sexes early in postmetamorphic development—potentially long before such behavior might serve an adaptive function. I place this result in the context of hypotheses regarding the development of learned versus non-learned behaviors, and in light of the potential for perception to be altered by physiological changes occurring concomitantly with ontogeny. Next, I describe a set of dynamic mate choice studies that highlight how decision-making in a relatively simple system is more flexible, and less stereotyped, than was previously assumed. Results here show that frogs temporally update their mate choice decisions in a moment-to-moment fashion as advertisement signals change in real time. By decomposing the decision-making process, I determine the stimulus parameters essential for commitment to an initial phonotactic approach. These studies are followed up by experiments that reveal a high level of individual variation in female choosiness during mate choice. Lastly, I describe a mate choice study that revealed categorical perception in frogs, the first “lower” vertebrate now known to exhibit a perceptual mode previously considered a hallmark of “higher” organisms. Collectively, I make the following arguments: (1) constraints on sensory systems play a larger role in shaping behavior than is generally appreciated, irrespective of the involvement of learning; (2) unstudied sources of variation may contribute significantly to the raw material for sexual selection; and (3) phonotaxis in anurans amphibians is not the simple, stereotyped behavior that has been suggested of it in the past.

Abstract  Mate choice studies most often examine female preferences based on population responses, thus potentially overlooking individual differences in behavior. Moreover, such studies typically use invariant stimulus conditions to infer preferences. By using population responses and static stimulus presentations, it is difficult to thoroughly understand the complexity of the mate choice process, including variation present between individuals. Here, we investigated phonotactic mate choice behavior in female tungara frogs (Physalaemus pustulosus) in response to temporally dynamic presentations of male advertisement calls. We tested females on repeated trials to examine individual differences and found considerable variation in the extent to which females update their mate choice decisions. Females in our study were bimodally distributed and thus broadly classified as either committed or uncommitted to an initial mate approach. We compared body condition measures of females differing behaviorally and determined that females with larger residual body masses were more committed to initial mate choices than less massive individuals, despite the fact that all females were in reproductive condition and field collected in amplexus. Our results suggest that anuran phonotaxis, once considered to be a highly stereotyped behavior, is more complex and variable than previously thought.

Abstract  We investigated the natural dynamics in a sexual signal that combines different call components and explored the role of call complexity in sexual selection using a neotropical frog. Male túngara frogs, Physalaemus pustulosus, facultatively add up to seven short, multiharmonic components (chucks) to the simple form of their calls (whines). Female túngara frogs are preferentially attracted to whines with chucks over whines without chucks, and males also call more in response to calls containing chucks. Because acoustic predators prefer complex calls, in the context of simple (no chucks) versus complex (any number of chucks) calls, the variably complex call appears to have evolved in response to the opposing selective forces of natural and sexual selection. There is no evidence, however, for the function of increasing the number of chucks within complex calls. We tested two aspects of increasing call complexity: natural patterns of use of call types in males and how both sexes respond to variation in multi-chuck calls. Males incrementally change call complexity by the addition or subtraction of a single chuck and usually do not produce more than two chucks. Variation in call complexity, for calls with at least one chuck, does not influence response calling in males or phonotaxis in females. Our results suggest that one reason for not increasing call complexity beyond a single chuck is the diminishing effectiveness on the responses of both sexes.