Estradiol induces sexual behavior in female túngara frogs

Steroid hormones play an important role in regulating vertebrate sexual behavior. In frogs and toads, injections of exogenous gonadotropins, which stimulate steroid hormone production, are often used to induce reproductive behavior, but steroid hormones alone are not always sufficient. To determine which hormonal conditions promote sexual behavior in female túngara frogs, we assessed the effect of hormone manipulation on the probability of phonotaxis behavior toward conspecific calls in post-reproductive females. We injected females with human chorionic gonadotropin (HCG), estradiol, estradiol plus progesterone, saline, or HCG plus fadrozole (an aromatase blocker) and tested their responses to mating calls. We found that injections of HCG, estradiol, and estradiol plus progesterone all increased phonotaxis behavior, whereas injections of saline or HCG plus fadrozole did not. Since injections of estradiol alone were effective at increasing phonotaxis behavior, we concluded that estradiol is sufficient for the expression of phonotaxis behavior. Next, to determine if estradiol-injected females display the same behavioral preferences as naturally breeding females, we compared mating call preferences of naturally breeding females to those of post-reproductive females injected with estradiol. We found that, when injected with estradiol, females show similar call preferences as naturally breeding females, although they were less likely to respond across multiple phonotaxis tests. Overall, our results suggest that estradiol is sufficient for the expression of sexual responses to mating calls in túngara frogs. To our knowledge, ours is the only study to find that estradiol alone is capable of promoting phonotaxis behavior in a frog.     

6‐hydroxydopamine lesions in anuran amphibians: A new model system for Parkinson's disease?

We investigated the effects of dopamine depletion on acoustically guided behavior of anurans by conducting phonotaxis experiments with female gray treefrogs (Hyla versicolor) before and 90 min after bilateral injections of 3, 6, or 12 μg 6‐hydroxydopamine (6‐OHDA) into the telencephalic ventricles. In experiments with one loudspeaker playing back a standard artificial mating call, we analyzed the effects of 6‐OHDA on phonotactic response time. In choice tests we measured the degree of distraction from the standard call (20 pulses/s) by three different variants with altered pulse‐rate (30/s, 40/s, 60/s). Five days after experiments, brains were immunostained for tyrosine hydroxylase. Labeled neurons were counted in the suprachiasmatic nucleus, posterior tuberculum, interpeduncular nucleus, and locus coeruleus, and correlation between neuronal numbers and behavioral scores was tested. Response times increased together with 6‐OHDA concentrations, which was mainly due to longer immobile periods before the animals started movement. In choice tests the most irrelevant stimulus (60/s) distracted 6‐OHDA injected females from the standard stimulus, while sham injected controls were undistracted. The number of catecholaminergic neurons decreased with increasing 6‐OHDA concentration in the suprachiasmatic nucleus, posterior tuberculum, and interpeduncular nucleus. The normalized number of immunoreactive neurons in the posterior tuberculum was positively correlated with phonotaxis scores in the one‐speaker test, demonstrating that motor deficits are a function of tubercular cell loss. We conclude that bilateral 6‐OHDA lesions in anuran amphibians cause motor (difficulty to start movements) as well as cognitive symptoms (higher distraction by irrelevant stimuli) that have also been described for human Parkinson patients. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 395–410, 2004     

Call recognition and female choice in a treefrog with a multicomponent call

Acoustic mating signals are typically species‐specific, and often additionally are subject to directional female preferences. Male executioner treefrogs, Dendropsophus carnifex, produce a multicomponent advertisement call composed of an introductory screech note followed by two or more click notes. Here, we tested (i) call recognition by comparing female directed phonotaxis towards individual and combined call components: screech vs. clicks vs. screech + clicks, (ii) female preferences for greater numbers of click notes and (iii) female preferences for faster call rates. The results demonstrated that screeches and clicks, presented either separately or together as a complete call, evoke similar female responses, suggesting that either note was sufficient to elicit a mate‐recognition response. Additionally, females preferred calls with greater numbers of click notes and with faster call rates. We interpret these results within the context of female mate selection in natural choruses.     

Pattern recognition and call preferences in treefrogs (Anura: Hylidae): a quantitative analysis using a no-choice paradigm

Studies of mate attraction have traditionally employed one of two experimental methods: choice tests in which female preferences from among two or more signals are tabulated, or single-stimulus tests in which the female response to one signal is quantified. Choice tests have long been preferred for examining mate attraction in anurans. Inspired by Wagner's (1998, Animal Behaviour,55, 1029-1042) discussion of the strengths and weaknesses of the two experimental approaches, we used a single-stimulus design to examine mate attraction in two sibling species of treefrogs (Hyla versicolor and H. chrysoscelis). We quantified female responses based upon the relative time required to approach signals varying in pulse rate, pulse rise time and pulse number. The data were used to generate response functions providing a quantitative measure of female attraction to the stimuli. Comparisons with data from choice experiments reveal broad similarities, as well as properties of female responses that had not been detected with choice tests. The results are discussed with regard to female selectivity for call parameters that are likely to mediate sexual selection and homospecific pairing.     

Phonotaxis to male’s calls embedded within a chorus by female gray treefrogs, Hyla versicolor

During the reproductive season, male Hyla versicolor produce advertisement calls to attract females. Females exhibit phonotaxis and approach the individual callers, resulting in amplexus. For frogs that call from dense choruses, the extent to which and the range from which a male’s advertisement call within a chorus can be heard by a receptive female leading to phonotaxis is unclear. We investigated females’ responses to natural choruses in the field and found that they were attracted and showed directed orientation to breeding choruses at distances up to 100 m. To assess the role of acoustic cues in the directed orientation, we conducted acoustic playback experiments in the laboratory using conspecific call and noise as stimuli, as well as chorus sounds (that contained calls from a focal male) recorded at various distances, all played at naturalistic intensities. Using two response metrics (females’ normalized response times and their phonotaxis trajectories) we found that, unlike the field experiments, females oriented and were attracted to chorus sounds from 1 to 32 m only, but not from >32 m, or to band-limited noise. Possible reasons for the observed difference in phonotaxis behavior in the two experimental conditions were discussed.     

Sound Pattern Recognition in Some North American Treefrogs (Anura: Hylidae): Implications for Mate Choice

SYNOPSIS. Playback experiments using synthetic sounds indicatethat gravid females of the gray treefrogs (Hyla versicolor andH. chrysoscelis) and the green treefrog (H. cinerea) selectivelyrespond to sounds on the basis of physical properties that showinter-individual variation in the calls of conspecific males.Although the specificity is adequate to differentiate amongthe signals of many males, this potential for mate choice maynot be fully realized in acoustically complex situations suchas natural breeding aggregations. Female selectivity in H. cinereawas less precise in four-speaker experiments than in two-speakerexperiments. An analysis of female preferences with respectto a call property correlated with body size was only partiallysuccessful in predicting the size distribution of successfulmales in a natural population. Finally, the existence of othersynchronously breeding species of frogs with similar calls mayimpose significant constraints on intraspecific mate choice,and female specificity with respect to some acoustic propertiesof mating calls is difficult to explain otherwise.     

Modality interactions alter the shape of acoustic mate preference functions in gray treefrogs

Sexual selection takes place in complex environments where females evaluating male mating signals are confronted with stimuli from multiple sources and modalities. The pattern of expression of female preferences may be influenced by interactions between modalities, changing the shape of female preference functions, and thus ultimately altering the selective landscape acting on male signal evolution. We tested the hypothesis that the responses of female gray treefrogs, Hyla versicolor, to acoustic male advertisement calls are affected by interactions with visual stimuli. We measured preference functions for several call traits under two experimental conditions: unimodal (only acoustic signals presented), and multimodal (acoustic signals presented along with a video‐animated calling male). We found that females were more responsive to multimodal stimulus presentations and, compared to unimodal playbacks, had weaker preferences for temporal call characteristics. We compared the preference functions obtained in these two treatments to the distribution of male call characteristics to make inferences on the strength and direction of selection expected to act on male calls. Modality interactions have the potential to influence the course of signal evolution and thus are an important consideration in sexual selection studies.     

Finding your mate at a cocktail party: frequency separation promotes auditory stream segregation of concurrent voices in multi-species frog choruses

Vocal communication in crowded social environments is a difficult problem for both humans and nonhuman animals. Yet many important social behaviors require listeners to detect, recognize, and discriminate among signals in a complex acoustic milieu comprising the overlapping signals of multiple individuals, often of multiple species. Humans exploit a relatively small number of acoustic cues to segregate overlapping voices (as well as other mixtures of concurrent sounds, like polyphonic music). By comparison, we know little about how nonhuman animals are adapted to solve similar communication problems. One important cue enabling source segregation in human speech communication is that of frequency separation between concurrent voices: differences in frequency promote perceptual segregation of overlapping voices into separate “auditory streams” that can be followed through time. In this study, we show that frequency separation (ΔF) also enables frogs to segregate concurrent vocalizations, such as those routinely encountered in mixed-species breeding choruses. We presented female gray treefrogs (Hyla chrysoscelis) with a pulsed target signal (simulating an attractive conspecific call) in the presence of a continuous stream of distractor pulses (simulating an overlapping, unattractive heterospecific call). When the ΔF between target and distractor was small (e.g., ≤3 semitones), females exhibited low levels of responsiveness, indicating a failure to recognize the target as an attractive signal when the distractor had a similar frequency. Subjects became increasingly more responsive to the target, as indicated by shorter latencies for phonotaxis, as the ΔF between target and distractor increased (e.g., ΔF = 6–12 semitones). These results support the conclusion that gray treefrogs, like humans, can exploit frequency separation as a perceptual cue to segregate concurrent voices in noisy social environments. The ability of these frogs to segregate concurrent voices based on frequency separation may involve ancient hearing mechanisms for source segregation shared with humans and other vertebrates.     

6-hydroxydopamine lesions in anuran amphibians: a new model system for Parkinson's disease?

We investigated the effects of dopamine depletion on acoustically guided behavior of anurans by conducting phonotaxis experiments with female gray treefrogs (Hyla versicolor) before and 90 min after bilateral injections of 3, 6, or 12 microg 6-hydroxydopamine (6-OHDA) into the telencephalic ventricles. In experiments with one loudspeaker playing back a standard artificial mating call, we analyzed the effects of 6-OHDA on phonotactic response time. In choice tests we measured the degree of distraction from the standard call (20 pulses/s) by three different variants with altered pulse-rate (30/s, 40/s, 60/s). Five days after experiments, brains were immunostained for tyrosine hydroxylase. Labeled neurons were counted in the suprachiasmatic nucleus, posterior tuberculum, interpeduncular nucleus, and locus coeruleus, and correlation between neuronal numbers and behavioral scores was tested. Response times increased together with 6-OHDA concentrations, which was mainly due to longer immobile periods before the animals started movement. In choice tests the most irrelevant stimulus (60/s) distracted 6-OHDA injected females from the standard stimulus, while sham injected controls were undistracted. The number of catecholaminergic neurons decreased with increasing 6-OHDA concentration in the suprachiasmatic nucleus, posterior tuberculum, and interpeduncular nucleus. The normalized number of immunoreactive neurons in the posterior tuberculum was positively correlated with phonotaxis scores in the one-speaker test, demonstrating that motor deficits are a function of tubercular cell loss. We conclude that bilateral 6-OHDA lesions in anuran amphibians cause motor (difficulty to start movements) as well as cognitive symptoms (higher distraction by irrelevant stimuli) that have also been described for human Parkinson patients.     

SPECIES RECOGNITION AND SEXUAL SELECTION AS A UNITARY PROBLEM IN ANIMAL COMMUNICATION

We investigated patterns of mating call preference and mating call recognition by examining phonotaxis of female túngara frogs, Physalaemus pustulosus, in response to conspecific and heterospecific calls. There are four results: females always prefer conspecific calls; most heterospecific calls do not elicit phonotaxis; some heterospecific calls do elicit phonotaxis and thus are effective mate recognition signals; and females prefer conspecific calls to which a component of a heterospecific call has been added to a normal conspecific call. We use these data to illustrate how concepts of species recognition and sexual selection can be understood in a unitary framework by comparing the distribution of signal traits to female preference functions.     

Bigger Is Not Always Better: Females Prefer Males of Mean Body Size in Philautus odontotarsus

Most species are believed to evolve larger body sizes over evolutionary time. Previous studies have suggested that sexual selection, through male-male competition and female choice, favors larger males. However, there is little evidence of selection against large size. The female serrate-legged small treefrogs (Philautus odontotarsus) must carry passive males from leks to breeding grounds over relatively long distances after amplexus to find a suitable place to lay eggs. The costs of large male size may therefore decrease mating success due to reduced agility and/or higher energy requirements. Thus, we hypothesized that selection would not favor larger males in P. odontotarsus. Females can assess male body size on the basis of the dominant frequency of male calls in frogs. To assess female P. odontotarsus preferences for a potential mate’s body size, male calls of high, average and low dominant frequency were played back to the females in phonotaxis experiments. Results showed that most females prefer the advertisement call with average dominant frequency. In addition, we compared the body mass distribution of amplectant males with that of single males in nature. The body masses of amplectant males are more narrowly distributed in the intermediate range than that of single males. The phonotaxis results and the data of actual female preferences in the field show that females strongly prefer potential mates of mean body sizes, consistent with the view that, in this species at least, larger males are not always perceived as better by females. In the present study, P. odontotarsus provides an example of an amphibian species in which large size does not have an advantage in mating success for males. Instead, our results provide evidences that stabilizing selection favors the optimal intermediate size of males.     

FORMS OF CALL OVERLAP AND THEIR IMPACT ON ADVERTISEMENT CALL ATTRACTIVENESS TO FEMALES OF THE GRAY TREEFROG,HYLA VERSICOLOR

ABSTRACT

Male gray Lreefrogs, Hyla versicolor, advertise for females in choruses in which calls often overlap. Previous research has demonstrated that such acoustic interference among conspecifics can impair the ability of a male to attract a mate. However, the stimulus set used in those studies was quite limited. Here we performed single-speaker and two-speaker tests of phonotaxis with female Hyla versicolor to explore the impact of different degrees and forms of call overlap on the relative attractiveness of male signals. We found that: (1) overlapped calls are recognized as acceptable signals by females, although they are less attractive than calls that are not overlapped; (2) the relative phase of the overlapping calls matters. When calls were time-shifted by 50% of their pulse-period (180 degree offset), attractiveness of overlapped calls was reduced relative to non-overlapped calls as much as it would be by a reduction in intensity of about 12 dB SPL or a drop in call duration of between one third to one half. A 90 degree phase offset was equivalent to a 3 dB drop in SPL; (3) partial call overlap may not impair call attractiveness if one third or less of the call is overlapped; (4) if the ending portion of a call is overlapped it may impair attractiveness less than if previous segments of a call are obscured; (5) if most (three fourths or more) of a male's calls are free of interference, overlap of his remaining calls does not significantly impair his attractiveness.     

Spatial hearing in Cope’s gray treefrog: I. Open and closed loop experiments on sound localization in the presence and absence of noise

The ability to reliably locate sound sources is critical to anurans, which navigate acoustically complex breeding choruses when choosing mates. Yet, the factors influencing sound localization performance in frogs remain largely unexplored. We applied two complementary methodologies, open and closed loop playback trials, to identify influences on localization abilities in Cope’s gray treefrog, Hyla chrysoscelis. We examined localization acuity and phonotaxis behavior of females in response to advertisement calls presented from 12 azimuthal angles, at two signal levels, in the presence and absence of noise, and at two noise levels. Orientation responses were consistent with precise localization of sound sources, rather than binary discrimination between sources on either side of the body (lateralization). Frogs were unable to discriminate between sounds arriving from forward and rearward directions, and accurate localization was limited to forward sound presentation angles. Within this region, sound presentation angle had little effect on localization acuity. The presence of noise and low signal-to-noise ratios also did not strongly impair localization ability in open loop trials, but females exhibited reduced phonotaxis performance consistent with impaired localization during closed loop trials. We discuss these results in light of previous work on spatial hearing in anurans.     

Do frog-eating bats perceptually bind the complex components of frog calls?

The mating calls of male túngara frogs, Physalaemus pustulosus, attract intended (conspecific females) and unintended (eavesdropping predators and parasites) receivers. The calls are complex, having two components: a frequency-modulated “whine” followed by 0–7 harmonic bursts or “chucks”. The whine is necessary and sufficient to elicit phonotaxis from females and the chuck enhances call attractiveness when it follows a whine. Although chucks are never made alone, females perceptually bind the whine and chuck when they are spatially separated. We tested whether an unintended receiver with independent evolution of phonotaxis, the frog-eating bat, Trachops cirrhosus, has converged with frogs in its auditory grouping of the call components. In contrast to frogs, bats approached chucks broadcast alone; when the chuck was spatially separated from the whine the bats preferentially approached the whine, and bats were sensitive to whine–chuck temporal sequence. This contrast suggests that although disparate taxa may be selected to respond to the same signals, different evolutionary histories, selective regimes, and neural and cognitive architectures may result in different weighting and grouping of signal components between generalist predators and conspecific mates.     

Elevated stress hormone diminishes the strength of female preferences for acoustic signals in the green treefrog

Mate selection can be stressful; time spent searching for mates can increase predation risk and/or decrease food consumption, resulting in elevated stress hormone levels. Both high predation risk and low food availability are often associated with increased variation in mate choice by females, but it is not clear whether stress hormone levels contribute to such variation in female behavior. We examined how the stress hormone corticosterone (CORT) affects female preferences for acoustic signals in the green treefrog, Hyla cinerea. Specifically, we assessed whether CORT administration affects female preferences for call rate — an acoustic feature that is typically under directional selection via mate choice by females in most anurans and other species that communicate using acoustic signals. Using a dual speaker playback paradigm, we show that females that were administered higher doses of CORT were less likely to choose male advertisement calls broadcast at high rates. Neither CORT dose nor level was related to the latency of female phonotactic responses, suggesting that elevated CORT does not influence the motivation to mate. Results were also not related to circulating sex steroids (i.e., progesterone, androgens or estradiol) that have traditionally been the focus of studies examining the hormonal basis for variation in female mate choice. Our results thus indicate that elevated CORT levels decrease the strength of female preferences for acoustic signals.     

How long do females really listen? Assessment time for female mate choice in the grey treefrog, Hyla versicolor

A satisfactory understanding of the process of mate choice in many species of anurans requires that we know how sensitive females are to the variation in male calls under natural conditions and what is the timescale or ‘window’ over which females compare potential mates. In natural choruses, grey treefrog females may sit near calling males for many minutes before approaching a particular individual to mate, whereas in laboratory-based phonotaxis tests, they may approach a speaker within 30 s of exposure to broadcast calls. Females prefer long versus short calls. To estimate ‘assessment time’ of females in nature, we broadcast calls from four pairs of 360-degree speakers suspended within screen cages at four locations at the edge of a pond. One speaker per pair presented calls of constant duration while the other speaker shifted between broadcasts of calls that were longer or shorter than the constant duration call. The time period over which this change in call duration occurred differed between the four venues. Laboratory-based choice tests indicated that females preferred call sources with variable numbers of pulses to those with constant numbers of pulses when the former had more total pulses per time window. Accordingly, we assigned the probabilities of field captures at the different speakers based on the summed pulses from the constant and cycling speakers within the possible assessment windows. These probabilities, together with the numbers of females captured at the speaker array over the breeding season, indicated that the most likely assessment time is close to 2 min.     

Reproductive stage and history affect the phonotactic preferences of female midwife toads, Alytes muletensis

We investigated whether the reproductive stage or reproductive history of females affects their preferences for male calls that differ in frequency. We repeatedly tested gravid female midwife toads prior to and immediately after mating, and during ovulation. When females were ovulating they were much more consistent in their choice of call than when they were gravid but not ovulating. When females had recently mated they still showed positive phonotaxis, but did not reliably discriminate between alternative calls. This study is the first to test gravid female anurans repeatedly at different stages in their reproductive cycle (ovulating or not; pre- and postmating). It highlights the need to treat with caution the results from population-based preference studies in which individuals are tested only once, and in which variation in reproductive state is not taken into account. Copyright 2000 The Association for the Study of Animal Behaviour.     

The Computational Mechanisms of Mate Choice

In many species, mating signals encode information important for both species recognition and mate quality assessment. I investigate how the computational mechanisms used by females to integrate the two sources of information can affect their mating decisions. First, I present a sequential analysis model of decision making based on a two‐component signal, in which the first component encodes information important for mate quality assessment and the second component for species recognition. When the components interact additively, the ability of females to discriminate between signals that differ in only the component important for mate‐quality assessment is the same independently of the value of the species‐recognition component. In contrast, when the components interact multiplicatively, discrimination in mate quality depends also on the species‐recognition component, which can either amplify or attenuate the perceived differences in mate quality. In the second part of the paper, I show results of a two‐choice phonotaxis experiment on female Italian treefrogs, Hyla intermedia, that confirm the model predictions by showing that directional preferences for call rate (important for mate‐quality assessment) are stronger when pulse‐rate and fundamental frequency (important for species recognition) are close to the preferred population mean than when they are either higher or lower than the mean.     

Phonotaxis in <Emphasis Type="Italic">Hyla versicolor</Emphasis> (Anura,Hylidae): the effect of absolute call amplitude

The influence of call amplitude on phonotaxis in female Hyla versicolor was studied using a no-choice paradigm. One set of experiments estimated effects of stimulus amplitude on phonotaxis toward a synthetic model of a conspecific call. The response strength increased with amplitude from the behavioral threshold (37–43 dB SPL) up to 79 dB SPL and then decreased at higher amplitudes. Females approached the loudspeaker with short walking bouts (1 s duration) occurring immediately after call presentations. Increase in response strength was attributed to an increasing proportion of calls that elicited such walking bouts, whereas the decrease at high amplitudes resulted from decreasing distance covered per bout. The quality of orientation remained constant for all above-threshold amplitudes. A second set of experiments tested the selectivity for interval duration and pulse duration at amplitudes of 55, 70, and 85 dB SPL. Selectivity for both parameters was similar at 70 and 85 dB SPL, but tended to increase at 55 dB SPL. The results suggest that selective phonotaxis in H. versicolor is not adapted for long-distance communication. This finding differs from those of comparable studies of acoustic insects.     

Factors affecting variation in the reproductive investment of female treefrogs,Hyla intermedia

In anurans, fecundity (clutch size) is the most important determinant of female reproductive success. We investigated three possible causes responsible for fecundity variation in female Italian treefrogs, Hyla intermedia, during four breeding seasons: (i) variation in morphological (body size and condition) and life-history (age) traits; (ii) variation in the tradeoff between the number and the size of eggs; (iii) seasonal effects and within-season differences in the timing of deposition. At the population level, we found no evidence for a tradeoff between the number and the size of eggs, because they both correlated positively with females’ body size. Conversely, neither age nor post-spawning body condition showed any effect on female reproductive investment. Independent of body size, we found no evidence for variation in reproductive effort among different breeding seasons, but strong evidence for a decrease of clutch size and an increase of egg size with the advancing of a breeding season. To test for the functional significance of the observed temporal variation in allocation strategy, we carried out a rearing experiment in semi-natural conditions on a random sample of ten clutches. The experiment showed a negative effect of clutch size and a positive effect of egg size on both tadpole growth and developmental rates, suggesting that reproductive investment, although constrained by body size, can be adjusted by females to the time of deposition to increase the chances of offspring survival.