Female frogs communicate with males through blinking.

Blinking is a behavior unique to animal taxa with motile eyelids, such as most amphibians and reptiles as well as all birds and mammals1. Eyelid movement has physiological functions, such as lubricating the cornea and washing away dust, but its potential signaling functions are not well understood1,2. The use of eyeblinks as a social signal is currently thought to be restricted to some primates, especially humans and their companion animals, but has not been verified in other taxa1,3,4. Here, through field observation and experiments, we demonstrate that female concave-eared torrent frogs (Odorrana tormota), which inhabit noisy streams, use eyeblinks to communicate with males to urge them to initiate amplexus for mating. Our findings reveal that eyeblinks may serve as a social signal in non-primate species.

Warmer temperature overrides the effects of antidepressants on amphibian metamorphosis and behavior.

Climate change can exacerbate the effects of environmental pollutants on aquatic organisms. Pollutants such as human antidepressants released from wastewater treatment plants have been shown to impact life-history traits of amphibians. We exposed tadpoles of the wood frog Lithobates sylvaticus to two temperatures (20 °C and 25 °C) and two antidepressants (fluoxetine and venlafaxine), and measured timing of metamorphosis, mass at metamorphosis, and two behaviors (startle response and percent motionless). Antidepressants significantly shortened time to metamorphosis at 20 °C, but not at 25 °C. At 25 °C, tadpoles metamorphosed significantly faster than those at 20 °C independent of antidepressant exposure. Venlafaxine reduced body mass at 25 °C, but not at 20 °C. Temperature and antidepressant exposure affected the percent of tadpoles showing a startle response. Tadpoles at 20 °C displayed significantly more responses than at 25 °C. Exposure to fluoxetine also increased the percent of tadpoles showing a startle response. Venlafaxine reduced the percent of motionless tadpoles at 25 °C but not at 20 °C. While our results showed that antidepressants can affect the timing of metamorphosis in tadpoles, warmer temperatures overrode these effects and caused a reduction in an important reaction behavior (startle response). Future studies should address how warmer global temperatures may exacerbate or negate the effects of environmental pollutants.

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.

Phenotypically plastic responses to freshwater salinization in larval amphibians: Induced tolerance and induced sensitivity.

Contamination of aquatic ecosystems is pervasive around the world and there has been a growing interest in understanding the ecological and evolutionary impacts. For contaminants such as pesticides, researchers are discovering widespread evolution of increased tolerance in target and non-target species and the role of phenotypic plasticity in facilitating this evolution. In contrast, we know much less about the evolution of tolerance in response to the increasing problem of freshwater salinization. In amphibians, recent studies have discovered that some populations from ponds with high salt pollution (from deicing road salts) have evolved higher tolerance. In this study, we examined whether populations of wood frog tadpoles (Rana sylvatica) possess rapid, inducible tolerance to salinity in a manner similar to their inducible tolerance to pesticides. Using newly hatched tadpoles from nine populations, we discovered that eight of the populations were able to alter their tolerance to salt. However, seven of the eight inducible populations experienced a higher sensitivity to salt while the eighth population experienced a higher tolerance to salt. Such inducible responses likely reflect the interplay of salt dynamics in the ponds, combined with the available genetic variation and selection intensity of each pond. This appears to be the first example of inducible salt tolerance in any animal and future studies should examine the generality of the response and how it may affect the evolution of tolerance to the global issue of freshwater salinization.

Developmental environment has lasting effects on amphibian post-metamorphic behavior and thermal physiology.

Environmental challenges early in development can result in complex phenotypic trade-offs and long-term effects on individual physiology, performance and behavior, with implications for disease and predation risk. We examined the effects of simulated pond drying and elevated water temperatures on development, growth, thermal physiology and behavior in a North American amphibian, Rana sphenocephala. Tadpoles were raised in outdoor mesocosms under warming and drying regimes based on projected climatic conditions in 2070. We predicted that amphibians experiencing the rapid pond drying and elevated pond temperatures associated with climate change would accelerate development, be smaller at metamorphosis and demonstrate long-term differences in physiology and exploratory behavior post-metamorphosis. Although both drying and warming accelerated development and reduced survival to metamorphosis, only drying resulted in smaller animals at metamorphosis. Around 1 month post-metamorphosis, animals from the control treatment jumped relatively farther at high temperatures in jumping trials. In addition, across all treatments, frogs with shorter larval periods had lower critical thermal minima and maxima. We also found that developing under warming and drying resulted in a less exploratory behavioral phenotype, and that drying resulted in higher selected temperatures in a thermal gradient. Furthermore, behavior predicted thermal preference, with less exploratory animals selecting higher temperatures. Our results underscore the multi-faceted effects of early developmental environments on behavioral and physiological phenotypes later in life. Thermal preference can influence disease risk through behavioral thermoregulation, and exploratory behavior may increase risk of predation or pathogen encounter. Thus, climatic stressors during development may mediate amphibian exposure and susceptibility to predators and pathogens into later life stages.

Male antiphonal calls and phonotaxis evoked by female courtship calls in the large odorous frog (Odorrana graminea).

Acoustic communication plays a vital role in frog reproduction. In most anuran species, long-distance sound communication is one-way from males to females; during the reproductive season, males produce species-specific advertisement calls to attract gravid females, and females are generally silent but perform phonotactic movements that lead to amplexus. One exception is the concave-eared torrent frog (Odorrana tormota). In this species, females produce courtship calls that elicit antiphonal vocalizations by males, followed by precise phonotactic movements. The large odorous frog O. graminea (previously Odorrana livida) in southern China is subject to the same environmental constraints as O. tormota, with which it is sympatric; it is unclear whether their sound communication is one-way or bidirectional. Here, we provide the first data on female O. graminea vocalizations and their functions. Using playbacks of female calls, we conducted acoustic behavioral experiments in the laboratory in response to which males emitted single- or multi-note antiphonal calls with a varying fundamental frequency. Moreover, they were attracted to female call playbacks, exhibiting precise phonotaxis. The female courtship call-male response interaction thus forms a duet between partners of a receptive pair. These results demonstrate that this unique communication system likely reflects an adaptation to an environment in which short-distance communication is at a premium given the high levels of ambient noise.

DPOAEs and tympanal membrane vibrations reveal adaptations of the sexually dimorphic ear of the concave-eared torrent frog, Odorrana tormota.

While most anuran species are highly vocal, few of them seem to be endowed with a complex call repertoire. Odorrana tormota, combines a remarkable vocalization complexity with auditory sensitivity over an extended spectral range spanning from audible to ultrasonic frequencies. This species is also exceptional for its ability to modify its middle ear tuning by closing the Eustachian tubes (ET). Using scanning laser Doppler vibrometry, the tympanal vibrations were measured to investigate if the tuning shift caused by the ET closure contributes to intraspecific acoustic communication. To gain insight into the inner ear frequency selectivity and sensitivity of this species, distortion product otoacoustic emissions were recorded at multiple frequency-level combinations. Our measurements of inner ear responses indicated that in O. tormota each sex is more sensitive to the frequencies of the other sex's vocalizations, female ears are more sensitive to 2-7 kHz, while male ears are more sensitive to 3-15 kHz. We also found that in both sexes the ET closure impacts the sensitivity of the middle and inner ear at frequencies used for communication with conspecifics. This study broadens our understanding of peripheral auditory mechanisms contributing to intraspecific acoustic communication in anurans.

Antipredator responses of Indosylvirana indica tadpoles do not match the level of predation risk.

Predators are the key regulators of prey populations in different environments. However, they are not always present but often come and go. Moreover, the level of predation risk varies based on habitat complexity, resource availability and other ecological factors. Hence, it is adaptive for prey animals to match their antipredator responses with the level of predation risk as such responses are costly to produce and maintain, and there exists a trade-off between fitness-enhancing and fitness-reducing activities. To test whether larval Indosylvirana adjust their antipredator responses based on the level of predation risk, we designed an experiment in which tadpoles of Indosylvirana indica were exposed to nil, low, moderate or high levels of predation risk to assess the effect of risk on growth, behaviour, morphology and life-history traits. We also determined the whole-body corticosterone levels to assess the physiological changes associated with the level of predation risk. Our results show that the growth rate of tadpoles experiencing varying levels of predation risk was similar although there was a trend towards a higher growth rate at moderate and high risks. Surprisingly, tadpoles experiencing predation risk did not reduce their activity. However, the activity of tadpoles experiencing differential predation risk was comparable. Similarly, the use of shelter was comparable among tadpole groups, with an overall higher level of activity in the afternoon compared to other times. Although a few morphological traits were different among tadpole groups, there was no trend or pattern. Moreover, these morphological alterations did not contribute to overall tadpole shape changes. Physiologically, tadpoles experiencing predation risk had significantly lower levels of corticosterone compared to those without risk. Interestingly, corticosterone titres among tadpoles facing varying levels of predation risk were similar. Metamorphic traits of individuals in the various predation risk groups were significantly different. Tadpoles experiencing moderate and high risks emerged at a larger size, and those experiencing the highest level of risk metamorphosed at the earliest. The results of our study thus show that the antipredator responses of larval I. indica do not match the level of predation risk, indicating that physiological and ecological constraints could limit the extent to which prey species respond to predation risk and its intensity.

Neuroendocrine correlates of juvenile amphibian behaviors across a latitudinal cline.

We assessed the macrogeographic and neuroendocrine correlates of behavioral variation exhibited by juveniles, an important life stage for dispersal, across the expansive range of the wood frog. By rearing animals from eggs in a common garden then using a novel environment test, we uniquely demonstrated differential expression of juvenile behaviors among 16 populations spanning 8° latitude. On the individual level, cluster analysis indicated three major behavior profiles and principal component analysis resolved four unique axes of behavior, including escape, foraging, food intake, feeding efficiency. We found that increased escape behavior was associated with lower adrenocorticotropic hormone (ACTH)-induced circulating corticosterone (CORT) levels, however, foraging and food intake behaviors were not associated with either resting or ACTH-induced CORT. At the population level, the expression of foraging behaviors increased with latitude while food intake behaviors declined with latitude, which raised several hypotheses of eco-evolutionary processes likely driving this variation. Given that these behaviors covary along the same ecological gradient as locally adapted developmental traits, genomic studies in this species could provide deep insights into how HPA/I activity is associated with the eco-evolutionary processes that structure intraspecific variation in morphology and behavior.

Effects of a Neonicotinoid Insecticide and Population Density on Behavior and Development of Wood Frogs (Rana sylvatica).

Amphibians have been facing global declines over the last decades from direct and indirect effects of anthropogenic activities. A contributor to declines is waterway contamination from agricultural runoffs of pesticides such as neonicotinoids. Beyond direct and indirect effects of the pesticide, few studies have investigated the possible interactions between neonicotinoids and natural environmental stressors across larval development, which could alter the strength and direction of observed neonicotinoid effects. The present study used a fully crossed design to investigate how a concentration of imidacloprid (a neonicotinoid; 10 µg/L) measured in surface waters interacted with low and high population densities (0.33 and 1 tadpole/L, respectively), an important environmental stressor, to influence behavior and development across metamorphosis in wood frogs (Rana sylvatica), known to breed in agricultural landscapes. Behaviors were measured in the absence and presence of predation cues using open-field tests at three distinct developmental stages, up to the metamorph stage. We found that imidacloprid did not interact with population density or independently affect behaviors in the absence of predation cues. However, individuals raised at high density compared with low density were more active at an early developmental stage but less active at metamorphic climax. Furthermore, both density and imidacloprid independently decreased the natural freezing response of tadpoles to predation cues. Finally, we found that distance traveled in the open-field test was weakly repeatable between aquatic stages but not repeatable across metamorphosis, a pattern that was not affected by treatments. The present study provides novel insights on the ecotoxicology of imidacloprid in the presence of a natural stressor, highlighting the importance of including behavioral assays and natural stressors in studies of amphibian ecotoxicology. Environ Toxicol Chem 2022;41:2968-2980. © 2022 SETAC.

Long-term sex-dependent inflammatory response of adult frogs to ammonium exposure during the larval stage.

Among others, the global change involves a worldwide increase in cropland area, with the concomitant rise in nitrogenous fertilizer supplementation and species range alterations, including parasites and pathogens. As most animals rely on their immune systems against these infectious agents, studying the potential effects of nitrogenous compounds on animal immune response is vital to understand their susceptibility to infections under these altered circumstances. Being subjected to an alarming process of global declines, amphibians are the object of particular attention, given their sensitivity to these compounds, especially to ammonium. Moreover, whereas adults can actively avoid polluted patches, larvae are confined within their waterbodies, thus exposed to contaminants in it. In this work, we test whether chronic exposure to a sublethal dose of ammonium during the larval stage of Pelophylax perezi frogs, released from all contamination after metamorphosis, leads to impaired inflammatory response to phytohemagglutinin in adults. We also test whether such a response differs between agrosystem individuals as compared with conspecifics from natural habitats. We found negative carryover effects of chronic exposure of larvae to ammonium on adult inflammatory response, which could imply a greater susceptibility to pathogens and parasites. However, this damage is only true for males, which, according to the immunocompetence handicap hypothesis, could be a consequence of a testosterone-triggered impairment of male immune function. In disagreement with our prediction, however, we detected no differences in the inflammatory response of agrosystem frogs to phytohemagglutinin as compared with natural habitat conspecifics.

Short-term responses of Rana arvalis tadpoles to pH and predator stress: adaptive divergence in behavioural and physiological plasticity?

Environmental stress is a major driver of ecological and evolutionary processes in nature. To cope with stress, organisms can adjust through phenotypic plasticity and/or adapt through genetic change. Here, we compared short-term behavioural (activity) and physiological (corticosterone levels, CORT) responses of Rana arvalis tadpoles from two divergent populations (acid origin, AOP, versus neutral origin, NOP) to acid and predator stress. Tadpoles were initially reared in benign conditions at pH 7 and then exposed to a combination of two pH (acid versus neutral) and two predator cue (predator cue versus no predator cue) treatments. We assessed behavioural activity within the first 15 min, and tissue CORT within 8 and 24 h of stress exposure. Both AOP and NOP tadpoles reduced their activity in acidic pH, but the response to the predator cue differed between the populations: AOP tadpoles increased whereas NOP tadpoles decreased their activity. The AOP and NOP tadpoles differed also in their CORT responses, with AOP being more responsive (CORT levels of NOP tadpoles did not differ statistically across treatments). After 8 h exposure, AOP tadpoles had elevated CORT levels in the acid-predator cue treatment and after 24 h exposure they had elevated CORT levels in all three stress treatments (relative to the benign neutral-no-cue treatment). These results suggest that adaptation to environmental acidification in R. arvalis is mediated, in part, via behavioural and hormonal plasticity.

DNA damage and repair responses to freezing and anoxia stresses in wood frogs, Rana sylvatica.

Wood frogs, Rana sylvatica, endure the freezing of ∼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also long-term anoxia due to cessation of heartbeat and breathing. Thawing restores perfusion but rapid reoxygenation can increase vulnerability to reactive oxygen species and induce oxidative damage. This study provides a first assessment of antioxidant capacity, DNA damage, and DNA repair responses comparing freeze/thaw and anoxia/reoxygenation in liver and skeletal muscle of wood frogs. Oxidation of guanine resides in DNA did not change under either stress but total antioxidant capacity rose in both tissues under anoxia. Relative expression of eight proteins involved in double-stranded break repair (Mre11, Rad50, phospho-p95, XLF, DNA ligase IV, XRCC4, Ku70, Rad51) were assessed in both tissues. Freezing suppressed Ku70 and Rad51 in liver and Rad51 in muscle but levels rose again after thawing. Anoxia exposure suppressed XLF, Ku70 and Rad51 proteins in muscle. However, in liver, anoxia exposure led to elevated Mre11, Ku70 and DNA ligase IV, the former two belonging to the MRN complex that binds DNA and marks sites of double stranded breaks (DSBs). Large increases in Mre11 and Ku70 expression suggested DSB damage in liver under anoxia but not during freezing, whereas muscle was resistant to DSB damage under both stresses. These data indicate that DNA damage is minimal during whole body freezing due to tissue and stress specific regulation of antioxidant capacity and DNA damage repair to preserve genomic integrity.

The morphological effects of artificial light at night on amphibian predators and prey are masked at the community level.

Artificial light at night (ALAN) is a pervasive pollutant that influences wildlife at both the individual and community level. In this study, we tested the individual-level effects of ALAN on three species of tadpole prey and their newt predators by measuring prey pigmentation and predator and prey mass. Then we evaluated whether the individual-level effects of ALAN on pigmentation and mass had cascading community-level effects by assessing the outcome of predator-prey interactions. We found that spring peepers exposed to ALAN were significantly darker than those reared under control conditions. Additionally, wood frogs reared in ALAN conditions were significantly smaller than those reared in control conditions. In contrast, Eastern newts collected earlier in the spring that were exposed to ALAN were significantly larger than controls while those collected later in the spring were not affected by ALAN, suggesting phenological differences in the effect of ALAN. To understand how changes in pigmentation and size due to ALAN influence predation rates, we ran predation assays in both ALAN-polluted and ALAN-free outdoor environments. After the predation assay, the size disparity in wood frogs reared in ALAN was eliminated such that there was no longer a treatment difference in wood frog size, likely due to size-selective predation. This demonstrates the beneficial nature of predators' selective pressure on prey populations. Lastly, despite individual-level effects of ALAN on pigmentation and mass, we did not detect cascading community-level effects on predation rates. Overall, this study highlights important species-level distinctions in the effects of ALAN. It also emphasizes the need to incorporate ecological complexity to understand the net impact of ALAN.

Lessons from nature: Leveraging the freeze-tolerant wood frog as a model to improve organ cryopreservation and biobanking.

The freeze-tolerant wood frog, Rana sylvatica, is one of the very few vertebrate species known to endure full body freezing in winter and thaw in early spring without any significant sign of damage. Once frozen, wood frogs show no cardiac or lung activity, brain function, or physical movement yet resume full physiological and biochemical functions within hours after thawing. The miraculous ability to tolerate such extreme stresses makes wood frogs an attractive model for identifying the molecular mechanisms that can promote freeze/thaw endurance. Recapitulating these pro-survival strategies in transplantable human cells and organs could improve viability post-thaw leading to better post-transplant outcomes, in addition to providing more time for adequate distribution of these transplantable materials across larger geographical areas. Indeed, several laboratories are beginning to mimic the pro-survival responses observed in wood frogs to preservation of human cells, tissues and organs and, to date, a few trials have been successful in extending preservation time prior to transplantation. In this review, we discuss the biology of the freeze-tolerant wood frog, current advances in biobanking based on these animals, and extend our discussion to future prospects for cryopreservation as an aid to regenerative medicine.

Development and validation of an eDNA protocol for monitoring endemic Asian spiny frogs in the Himalayan region of Pakistan.

Wildlife monitoring programs are instrumental for the assessment of species, habitat status, and for the management of factors affecting them. This is particularly important for species found in freshwater ecosystems, such as amphibians, as they have higher estimated extinction rates than terrestrial species. We developed and validated two species-specific environmental DNA (eDNA) protocols and applied them in the field to detect the Hazara Torrent Frog (Allopaa hazarensis) and Murree Hills Frog (Nanorana vicina). Additionally, we compared eDNA surveys with visual encounter surveys and estimated site occupancy. eDNA surveys resulted in higher occurrence probabilities for both A. hazarensis and N. vicina than for visual encounter surveys. Detection probability using eDNA was greater for both species, particularly for A. hazarensis. The top-ranked detection model for visual encounter surveys included effects of both year and temperature on both species, and the top-ranked occupancy model included effects of elevation and year. The top-ranked detection model for eDNA data was the null model, and the top-ranked occupancy model included effects of elevation, year, and wetland type. To our knowledge, this is the first time an eDNA survey has been used to monitor amphibian species in the Himalayan region.

Naive poison frog tadpoles use bi-modal cues to avoid insect predators but not heterospecific predatory tadpoles.

For animals to survive until reproduction, it is crucial that juveniles successfully detect potential predators and respond with appropriate behavior. The recognition of cues originating from predators can be innate or learned. Cues of various modalities might be used alone or in multi-modal combinations to detect and distinguish predators but studies investigating multi-modal integration in predator avoidance are scarce. Here, we used wild, naive tadpoles of the Neotropical poison frog Allobates femoralis ( Boulenger, 1884) to test their reaction to cues with two modalities from two different sympatrically occurring potential predators: heterospecific predatory Dendrobates tinctorius tadpoles and dragonfly larvae. We presented A. femoralis tadpoles with olfactory or visual cues, or a combination of the two, and compared their reaction to a water control in a between-individual design. In our trials, A. femoralis tadpoles reacted to multi-modal stimuli (a combination of visual and chemical information) originating from dragonfly larvae with avoidance but showed no reaction to uni-modal cues or cues from heterospecific tadpoles. In addition, visual cues from conspecifics increased swimming activity while cues from predators had no effect on tadpole activity. Our results show that A. femoralis tadpoles can innately recognize some predators and probably need both visual and chemical information to effectively avoid them. This is the first study looking at anti-predator behavior in poison frog tadpoles. We discuss how parental care might influence the expression of predator avoidance responses in tadpoles.

Evaluation of antimicrobial and anticancer activities of three peptides identified from the skin secretion of Hylarana latouchii.

The skins of frogs of the family Ranidae are particularly rich sources of biologically active peptides, among which antimicrobial peptides (AMPs) constitute the major portion. Some of these have attracted the interest of researchers because they possess both antimicrobial and anticancer activities. In this study, with 'shotgun' cloning and MS/MS fragmentation, three AMPs, homologues of family brevinin-1 (brevinin-1HL), and temporin (temporin-HLa and temporin-HLb), were discovered from the skin secretion of the broad-folded frog, Hylarana latouchii. They exhibited various degrees of antimicrobial and antibiofilm activities against test microorganisms and hemolysis on horse erythrocytes. It was found that they could induce bacteria death through disrupting cell membranes and binding to bacterial DNA. In addition, they also showed different potencies towards human cancer cell lines. The secondary structure and physicochemical properties of each peptide were investigated to preliminarily reveal their structure-activity relationships. Circular dichroism spectrometry showed that they all adopted a canonical α-helical conformation in membrane-mimetic solvents. Notably, the prepropeptide of brevinin-1HL from H. latouchii was highly identical to that of brevinin-1GHd from Hylarana guentheri, indicating a close relationship between these two species. Accordingly, this study provides candidates for the design of novel anti-infective and antineoplastic agents to fight multidrug-resistant bacteria and malignant tumors and also offers additional clues for the taxonomy of ranid frogs.

Influence of octylphenol on gene expression of gonadotropins and their receptors, testicular structure and mating behavior of male Rana chensinensis.

In the present study, responses of the Chinese brown frog (Rana chensinensis) to exposure to different doses and duration of Octyphenol (OP) which degraded from alkylphenol ethoxylates (APEs) were characterized during the adult periods. The effects of OP on growth, development and reproduction and the expression of genes in gonad were investigated. The expression levels of fshß, lhß, fshr and lhr had significant differences as the exposure time increased. The pathological and morphological changes were also observed in the OP treatments. Furthermore, the number of TUNEL positive cells and the TUNEL index was elevated after exposed to OP. Besides that, OP treatment could influence its mating behavior and reduce the fertilization rates. Taken together, these results indicated that OP disrupt sex steroid signaling, normal development of spermatogenesis, courtship behavior of male frogs and decline fertilization rate in R. chensinensis.

Androgen Receptor Modulates Multimodal Displays in the Bornean Rock Frog (Staurois parvus).

Multimodal communication is common in the animal kingdom. It occurs when animals display by stimulating two or more receiver sensory systems, and often arises when selection favors multiple ways to send messages to conspecifics. Mechanisms of multimodal display behavior are poorly understood, particularly with respect to how animals coordinate the production of different signals. One important question is whether all components in a multimodal display share an underlying physiological basis, or whether different components are regulated independently. We investigated the influence of androgen receptors (ARs) on the production of both visual and vocal signal components in the multimodal display repertoire of the Bornean rock frog (Staurois parvus). To assess the role of AR in signal production, we treated reproductively active adult males with the antiandrogen flutamide (FLUT) and measured the performance of each component signal in the multimodal display. Our results show that blocking AR inhibited the production of multiple visual signals, including a conspicuous visual signal known as the "foot flag," which is produced by rotating the hind limb above the body. However, FLUT treatment caused no measurable change in vocal signaling behavior, or in the frequency or fine temporal properties of males' calls. Our study, therefore, suggests that activation of AR is not a physiological prerequisite to the coordination of multiple signals, in that it either does not regulate all signaling behaviors in a male's display repertoire or it does so only in a context-dependent manner.