Male Frogs Research Articles

Anuran gender identification by fecal steroid analysis

AbstractThis study tested the hypothesis that steroid hormone metabolites can be measured in anuran feces and their concentrations used to identify the sex of adults. Fecal samples from American toads, Bufo americanus, and boreal toads, B. boreas boreas, were extracted using ethyl acetate, and the concentrations of estradiol, progesterone and testosterone metabolites were measured by enzyme immunoassays with antibodies commonly used to evaluate steroid hormone concentrations in mammalian species. In American toads, mean testosterone metabolite concentrations (P<0.05) between males (224.3±15.5 ng/g feces) and females (80.7±10.6 ng/g), but estradiol and progesterone metabolite concentrations did not. In contrast, estradiol immunoreactivity differed (P<0.05) between male (19.0±1.8 ng/g) and female (48.3±6.3 ng/g) boreal toads. Progesterone and testosterone metabolite concentrations did not differ. Fecal hormone metabolite analysis offers a promising noninvasive approach to gender identification in anuran amphibians. However, the group of metabolites differentiating gender may not be consistent among species. Zoo Biol 0:1–12, 2005. © 2005 Wiley‐Liss, Inc.

Elevated corticosterone levels elicit non-calling mating tactics in male toads independently of changes in circulating androgens

Competition among males for a limited number of females may result in the expression of condition-dependent alternative mating tactics. In such cases, decision rules mediating mating tactic expression are likely to be influenced by a male's external as well as internal environment. For example, experimental studies with anuran amphibians (frogs and toads) indicate that changes in the social-acoustic environment alter the probability that an individual adopts a calling versus non-calling “satellite” mating tactic. However, there is considerable variation in the behavioral responses of individuals in such studies, suggesting that physiological differences among individuals play an important role in tactic expression. For instance, recent models predict that natural elevation in adrenal glucocorticoids during vocal production alters androgen production and/or neural activity to mediate transitions between reproductive tactic expression in anuran amphibians. Using corticosteroid injections, we show that elevation in circulating corticosterone levels significantly increase the probability that free-ranging male Great Plains toads ( Bufo cognatus) and Woodhouse's toads ( B. woodhousii) adopt a non-calling satellite tactic. Corticosterone-induced behavioral transitions occurred rapidly (<1 h) and independently of fluctuations in circulating androgen levels, suggesting a direct effect of glucocorticoids on brain centers controlling vocal production. We discuss our findings in the context of behavioral studies that alter the social-acoustic environment to examine its influence on tactic expression.

Potential ecotoxic effects of polychlorinated biphenyls on Xenopus laevis

We examined potential ecotoxic effects of polychlorinated biphenyl (PCB)3, PCB5, Aroclor 1254, and Aroclor 1242 on Xenopus laevis. Tadpoles were exposed to PCBs from stage 46/47 (system of Nieuwkoop and Faber) to the completion of metamorphosis. We demonstrated, to our knowledge for the first time, forelimb malformations caused by PCBs (malformation rate, > 70%). The malformed forelimbs were fixed in the adduction-backward rotation position and could not move. Therefore, malformed male frogs were destined to have no offspring, because they could not grasp the females with their forelimbs to mate. Alcian blue-alizarin red double-staining indicated that the forelimb malformation resulted from the shoulder abnormality. Compared with the normal shoulder joint, the proximal humerus with the humerus inter-rotated 90 degrees in the abnormal shoulder joint. Moreover, testes from more than a third of male frogs with exposed to PCBs exhibited feminization to different degrees at gross morphology and histology, with fewer or abnormal spermatogonia and oocytes. Gonadal abnormalities would lead directly to reproductive dysfunction and population decline. These results suggest that PCBs have potentially ecotoxic effects on amphibian populations. We infer that PCBs could play roles in amphibian malformations and population declines, at least at sites that are polluted heavily with PCBs.

Vocally mediated social recognition in anurans

Anuran amphibians (frogs and toads) are among the most vocal of vertebrates and have long served as model systems for investigating the mechanisms and evolution of acoustic communication. Compared to higher vertebrates, however, the role of cognition in anuran communication has received less attention, at least in part due to the lack of evidence that juvenile anurans learn to produce signals or associate them with particular social contexts. Recent studies of social recognition in two anuran families indicate that territorial male frogs in some species are able to learn about and recognize the individually distinctive properties of the calls of nearby neighbors. For example, male bullfrogs (ranidae) learn about the pitch of a neighbor’s vocalizations (an individually distinct voice property) and associate a familiar pitch with the location of the neighbor’s territory. As in songbirds, this form of vocally mediated social recognition allows territory holders to direct low levels of aggression toward well-established neighbors, while maintaining a readiness to respond aggressively to more threatening strangers that may attempt a territory takeover. A brief review of currently available data will be used to illustrate how anurans can serve as model systems for investigating the role of cognition in acoustic communication.

A new record for PBDEs in people | POPs treaty targets further chemicals | Canada's research funding system works | Do male frogs naturally have female traits? | Are low levels of lead in water risky?

ADVERTISEMENT RETURN TO ISSUENewsNEXTA new record for PBDEs in people | POPs treaty targets further chemicals | Canada's research funding system works | Do male frogs naturally have female traits? | Are low levels of lead in water risky?Kellyn S. Betts, Janet Pelley, and Rebecca RennerCite this: Environ. Sci. Technol. 2005, 39, 14, 296A–298APublication Date (Web):July 15, 2005Publication History Published online15 July 2005Published inissue 1 July 2005https://doi.org/10.1021/es0533085RIGHTS & PERMISSIONSArticle Views303Altmetric-Citations2LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (128 KB) Get e-AlertsSUBJECTS:Ethers Get e-Alerts

Historical perspective: Hormonal regulation of behaviors in amphibians

This review focuses on research into the hormonal control of behaviors in amphibians that was conducted prior to the 21st century. Most advances in this field come from studies of a limited number of species and investigations into the hormonal mechanisms that regulate reproductive behaviors in male frogs and salamanders. From this earlier research, we highlight five main generalizations or conclusions. (1) Based on studies of vocalization behaviors in anurans, testicular androgens induce developmental changes in cartilage and muscles fibers in the larynx and thereby masculinize peripheral structures that influence the properties of advertisement calls by males. (2) Gonadal steroid hormones act to enhance reproductive behaviors in adult amphibians, but causal relationships are not as well established in amphibians as in birds and mammals. Research into the relationships between testicular androgens and male behaviors, mainly using castration/steroid treatment studies, generally supports the conclusion that androgens are necessary but not sufficient to enhance male behaviors. (3) Prolactin acts synergistically with androgens and induces reproductive development, sexual behaviors, and pheromone production. This interaction between prolactin and gonadal steroids helps to explain why androgens alone sometimes fail to stimulate amphibian behaviors. (4) Vasotocin also plays an important role and enhances specific types of behaviors in amphibians (frog calling, receptivity in female frogs, amplectic clasping in newts, and non-clasping courtship behaviors). Gonadal steroids typically act to maintain behavioral responses to vasotocin. Vasotocin modulates behavioral responses, at least in part, by acting within the brain on sensory pathways that detect sexual stimuli and on motor pathways that control behavioral responses. (5) Corticosterone acts as a potent and rapid suppressor of reproductive behaviors during periods of acute stress. These rapid stress-induced changes in behaviors use non-genomic mechanisms and membrane-associated corticosterone receptors.

Aspects of the population ecology of the Green and golden bell frogLitoria aureaat the northern end of its range

Development of appropriate conservation programs for threatened species requires a basic understanding of their population ecology. We studied a population of the Green and golden bell frog Litoria aurea in the far north of its range. This population occupies a coastal lagoon within Yuraygir National Park. Permanent tagging of frogs was conducted between August 1998 and March 2003. Population estimation based on the mark and recapture of frogs in 1998-99 suggested that the population contained at least 100 adult male frogs.Approximately 75 male bell frogs called from around the lagoon on one night in November 200 I .The number of females captured in any year was low and it is unclear whether this is due to low detectability associated with irregular use of breeding sites or a highly skewed sex ratio. More than 60% of tagged male frogs were recaptured across one active season, but only two were recaptured outside the season of initial capture.This suggests that few male frogs live longer than about 2 years as adults. Breeding was erratic over a 6-year period and only one major metamorphosis event was observed. It appears that bell frogs may have greater breeding success in ephemeral ponds forming away from the lagoon, though there is a risk that these ponds may not persist for tadpoles to complete development. Frogs in this northern population were predominantly brown in colour, suggesting some morphological and genetic differentiation from southern populations in which most frogs are predominantly green. Further study of this important population is needed to more adequately determine the size of the female component of the population, and to better understand the limits on successful breeding.These issues will influence our understanding of the viability of the population, and therefore our ability to devise appropriate management actions. This study provides the first published account of aspects of the population ecology of the Green and golden bell frog in relatively undisturbed natural habitat.

The effect of perceived intruder proximity and resident body size on the aggressive responses of male green frogs, Rana clamitans (Anura: Ranidae)

We examined variation in aggressive responses within and among individuals in the green frog, Rana clamitans. We tested the hypothesis that resident males respond in a graded fashion to changes in perceived intruder proximity (stimulus intensity). We also investigated how response level varied with responder body size. We found that green frogs differentially alter aggressive responses (increasing movement and calling rates while decreasing the duration and dominant frequency of their calls) with an increase in stimulus intensity. Body size did not appear to be a significant influence on most response levels. We suggest that aggressive responses are graded, and advertisement and aggressive calls represent opposite ends of a continuum. Green frogs do not exhibit clearly defined thresholds for responses like those reported for some other frogs. By using graded responses rather than discrete “all-or-nothing” responses, males may reduce the potential costs of aggressive encounters.

Neurodistribution of Androgen Receptor Immunoreactivity in the Male Frog, Rana esculenta

Sexual behavior in vertebrates depends on the cyclic release of steroids and their binding to the brain receptors. Previously, we demonstrated the presence of specific binding of (3)H-testosterone and staining with PG-21 in the brain of the adult male frog, Rana esculenta. Here, we report our further receptor characterization using an anti-androgen receptor antiserum, PG-21, and the androgen site of action in frog brain. Nuclei, which contained cells labeled for the androgen receptor (AR), were mainly identified in the olfactory bulbs, preoptic-septal region, infundibulum, amygdala, thalamus, tectum, torus semicircularis, and medulla. The neuroanatomical AR staining appears similar to that in other lower vertebrates.

Testosterone secretion and pharmacological spermatozoal recovery in the cane toad ( Bufo marinus)

The cane toad ( Bufo marinus) was used as a model to study male anuran reproductive endocrinology and to develop a protocol for non-invasive sperm recovery. Circulating testosterone concentrations in 6-hourly samples did not vary significantly ( P < 0.05) over a 24 h period although there was a tendency ( P = 0.06) for testosterone to be elevated at 19:00 h relative to other times of the day, which may be related to the nocturnal activity pattern of this species. Testosterone secretion after intraperitoneal (IP) injection of either a GnRH agonist (5 μg IP) or hCG (1000 IU) was also examined. While the GnRH agonist did not produce a significant increase above basal plasma testosterone (0.29, 95% C.I. of 0.05–1.10 ng/ml), injection of hCG resulted in an increase ( P < 0.01) of plasma testosterone with peak concentrations at approximately 120 min (4.17, 95% C.I. of 2.69–7.44 ng/ml) after injection. Non-invasive pharmaceutical sperm recovery was attempted following IP injection of graded doses of GnRH agonist, hCG or FSH. Urine was collected at 3, 6 and 12 h after treatment to assess sperm quality and quantity. The optimal protocol for sperm recovery in cane toads was injection of either 1000 or 2000 IU hCG; there was no significant difference in the quality of the spermic urine samples obtained using either dose of hCG or with respect to collection time. The findings indicated that hCG can be used to assess testicular steroidogenic status and also to induce sperm recovery in the cane toad. The hCG protocols developed in this study will have application in studies on the reproductive biology of rare and endangered male anurans.

THRESHOLDS FOR EVOKED VOCAL RESPONSES OF EUPSOPHUS EMILIOPUGINI (AMPHIBIA, LEPTODACTYLIDAE)

Thresholds for evoked advertisement calling for 18 males were measured in a temperate forest in southern Chile, in order to relate this behavior to the intensities of neighbors' calls that males of this species encounter in nature. Stimuli used were bouts of natural single-note advertisement calls played back at increasing intensity in steps of 6–12 dB. Male frogs responded with single-note advertisement calls to stimulus bouts at average sound levels of about 60 dB peak SPL (range: 39–85 dB peak SPL), measured at the subjects' position. Frogs increased their call rates in response to increasing stimulus intensity, reaching a plateau at about 24 dB above threshold. At intensities above threshold, 11 frogs also responded with double-note calls, and two of these frogs occasionally gave triple- and four-note calls. The average thresholds for advertisement calling are low enough so that males respond to their nearest neighbors in low-density aggregations, thereby sustaining the chorusing activity.

The effects of reactive oxygen species on amphibian aging

To clarify the role of reactive oxygen species (ROS) in the aging process of amphibians, antioxidant enzyme activity and indexes of ROS damage were investigated biochemically using the livers of 3- and 10-year-old Rana nigromaculata frog males and females. Findings revealed no significant difference in survival rate between males and females. Antioxidant enzyme activity displayed an age-related decline. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activity in 10-year-old liver decreased 40–80% from 3-year-old liver levels. In contrast, urate oxidase activity in the 10-year-old liver increased more than 200% from 3-year-old liver levels. At the same time levels of ROS damage, including the concentration of inorganic peroxide and thiobarbituric acid reactive substances (TBARS), greatly increased with age. Liver catalase from 10-year-old frogs proved to be more susceptible to aminotriazole and urea, losing approximately 80% of its original activity after 30 min of treatment. It seems likely that liver catalase in older frogs has diverged from liver catalase in younger frogs through oxidative modification. These findings suggest that a decrease in the activity of antioxidant enzymes over time results in increased levels of ROS damage in the livers of older frogs.

Effects of atrazine on metamorphosis, growth, laryngeal and gonadal development, aromatase activity, and sex steroid concentrations in Xenopus laevis

African clawed frogs ( Xenopus laevis) were exposed to one of eight nominal waterborne concentrations including 0, 0.1, 1.0, 10, or 25 μg/L atrazine, 0.005% ethanol (EtOH), or 0.1 mg/L estradiol (E2) or dihydrotestosterone (DHT) containing 0.005% EtOH. Frogs were exposed from 72 h posthatch until 2–3 months postmetamorphosis via a 3-day static renewal exposure regimen. Atrazine at concentrations between 0.1 and 25 μg/L did not significantly affect mortality, growth, gonad development, laryngeal muscle size, or aromatase activity in juvenile X. laevis. Male frogs exposed to 1.0 μg/L atrazine had lower E2 levels compared to controls, but this response was not consistent among other concentrations of atrazine. Male and female frogs exposed to DHT had larger laryngeal dilator muscle areas compared to controls. E2-exposed female frogs had decreased gonadal aromatase activity, and E2-exposed male frogs had statistically greater plasma concentrations of E2 compared to controls.

Effects of Nonylphenol and Triclosan on Production of Plasma Vitellogenin and Testosterone in Male South African Clawed Frogs (Xenopus laevis)

We investigated the effects of nonylphenol (NP) and triclosan (TCS) on production of vitellogenin (Vg), testosterone (T), and hepatic cytochrome P450 1A and 2B activities in male South African clawed frogs (Xenopus laevis). In a 14-d waterborne exposure test, no significant differences in the level of plasma Vg synthesis in male frogs were observed among the control, 10, 50, and 100 microg/l NP and 20, 100, and 200 microg/l TCS treatment groups. Intraperitoneal injection of male frogs with 2, 20, and 200 microg/g body weight NP resulted in no significant differences in plasma Vg levels among the control and all treatment groups. However, the levels of plasma Vg in all TCS treatment groups (intraperitoneal injection of 4, 40, and 400 microg/g body weight) were lower than that in the solvent control group, and male frogs injected with high doses of NP or TCS had lower T levels than the control group. No significant differences in hepatic cytochrome P450 1A and 2B activities were observed among the all treatment groups. Male frogs injected with 20 microg/g body weight of estradiol-17beta had significantly higher plasma Vg levels than the control group. These results suggest that profiles of plasma Vg and T production in male Xenopus laevis could be useful biomarkers for detecting hormonally active agents.

Assessment of laryngeal muscle and testicular cell types in Xenopus laevis (Anura Pipidae) inhabiting maize and non‐maize growing areas of South Africa

We tested the hypothesis that adult African clawed frogs (Xenopus laevis) inhabiting water bodies in maize‐growing areas (MGA) of South Africa would exhibit differences in testicular structure compared to frogs from water bodies in non‐maize‐growing areas (NMGA) in the same locale. Adults of both sexes were collected during the autumn of 2002 in South Africa, and stereological analytical techniques were used to quantify the distribution of testicular cell types. In addition, total laryngeal mass was used as a gauge of secondary sex differences in animals from MGA and NMGA study sites. Evaluation of the total laryngeal mass revealed that there were no statistically significant differences between X. laevis of the same sex from the NMGA and MGA sites. Mean percent fractional‐volume values for seminiferous tubule distribution of testicular cell types of mature X. laevis, ranged from 3–4% for spermatogonia, 26–28% for spermatocytes, 54–57% for spermatozoa, and 14–15% for other cells types. The mean percent volume for blood vessels ranged from 0.3–0.4%. These values did not differ significantly between frogs from NMGA and MGA areas. Collectively, these data demonstrated no differences in gonadal and laryngeal development in X. laevis collected in South Africa from MGA and NMGA areas and that there is little evidence for an effect of agricultural chemicals used in maize production functioning as endocrine disrupters in this species. Screening of X. laevis testes revealed a small incidence of Stage 1 testicular oocytes in adult male frogs collected from the NMGA (3%) and MGA (2%).

Chorus tenure and estimates of population size of male European tree frogs Hyla arborea: implications for conservation

AbstractChorusing male anurans typically spend only a part of the breeding season calling although chorus tenure is often the best predictor of mating success. We determined the number of nights males attended a chorus (chorus tenure) and its influence on mating success in the European tree frog, Hyla arborea. The median chorus tenure was 7.5 nights out of a study season of 38 nights. Males that spent more than two nights in the chorus were present for an average of 47% of the nights between their first and last night in the chorus. Minimum daily temperature, ambient temperature at initiation of calling, and daily rainfall explained 37.8% of the variance in male attendance. Twenty-five males were calling on the night of peak activity, a fraction of the 44 males marked. This suggests that estimates of male population size based on peak activity, widely used by conservation biologists, are inaccurate. We suggest that, when mark-recapture methods cannot be used, male population size be calculated by using a regression model based on the peak number of calling males that can be further developed as more data accumulates.

Characterization of a peptide from skin secretions of male specimens of the frog, Leptodactylus fallax that stimulates aggression in male frogs

During the breeding season of the mountain chicken frog Leptodactylus fallax, fighting between males results in the emergence of dominant animals that subsequently attract females to nesting sites. A peptide, termed Leptodactylus aggression-stimulating peptide (LASP), was isolated from norepinephrine-stimulated skin secretions from male specimens of L. fallax that was not present in skin secretions obtained from females. The primary structure of the peptide was established as: Gly-Leu-Trp-Asp-Asp-Leu-Lys-Ala-Ala-Ala-Lys-Lys-Val-Val-Ser-Ser-Leu-Ala-Ser-Ala-Ala-Ile-Glu-Lys-Leu NH 2. LASP had no pheromone-like action on females but had a chemoattractive effect on males and stimulated aggressive behaviors, such as rearing and leaping. It is suggested that this peptide may play an important role in initiating the competitive male–male interactions that are associated with the onset of reproductive behavior in L. fallax.

Plant Selection and Seasonal Patterns of Vocal Activity in Two Populations of the Bromeligen Treefrog Scinax perpusillus (Anura, Hylidae)

An interesting case of reproductive specialization among anurans is found in bromeligen species that have reproductive cycles associated with bromeliads. In these species, male reproductive activity might be related not only to macroclimatic patterns but also to the availability of suitable microhabitats in bromeliads. This research focuses on one bromeligen species from Southeastern Brazil, Scinax perpusillus, that is territorial and uses bromeliads as retreat and oviposition sites. Two populations were studied (Intervales State Park and Boracéia Biological Station, both in the state of São Paulo, Brazil). Available bromeliads vary substantially in morphology and position in the forest. Thus, we examined whether male frogs (1) select plants with specific eco-morphological traits, (2) call at specific time periods within the reproductive season, and (3) call more actively under certain temperature and humidity conditions. Patterns of male calling activity within the reproductive season varied between years and between populations, but calling was not more intense during warmer or more humid periods. It seems, then, that subtle patterns of phenology might vary significantly between populations and across years. Bromeliad choice was nonrandom. Males chose bromeliads that were larger, clustered, closer to the ground, and had higher reservoir pH, features that probably reflect the quality of the plants as oviposition sites and that might confer social advantages by reducing conflict with neighboring males.

Using Chorus-Size Ranks from Call Surveys to Estimate Reproductive Activity of the Wood Frog (Rana sylvatica)

Call surveys are a relatively new and efficient technique for detecting the presence of breeding male anurans. Using data from multiple surveys of breeding choruses of Wood Frogs (Rana sylvatica) combined with counts of egg masses on ponds in west-central Alberta we determined (1) the relationship between ranks of chorus size and total number of egg masses in ponds, and (2) number of breeding males in standard chorus-size ranks (1, 2 and 3). Estimates for Rank 3 choruses were based on a formula with number of egg masses present per pond and a fixed male to female ratio of 2:1 calculated from the literature. Calling males were recorded from all ponds that had evidence of female reproductive activity (i.e., egg masses). Generalized linear models suggested that ranks were positively and linearly correlated with the number of egg masses in a pond. In addition, call data from only the second of four sampling periods (each 3–6 days) significantly predicted number of egg masses in ponds, suggesting that timing is important when surveying calling wood frogs. The mean number of chorusing males per rank did not correspond to aural ranks of calling intensity: Rank 1 = 1.3 males, Rank 2 = 3.7 males, and Rank 3 = 118 males. We recommend similar assessments for other widely distributed species to improve our ability to detect and interpret habitat-use patterns and population trends of amphibians through monitoring programs.

Neuronal responses are differentially affected by the polarity of tectal DC stimulation in the toad Bufo bufo

Male toads were tested behaviourally for their prey catching responses to worm-like stimuli before being prepared for visual unit and slow potential shift (SPS) recording from the optic tectum. The neuronal responses of toads to a prey-like visual stimulus reflected their motivational tendency prior to operations. One second of DC stimulation to the tectum was followed by an SPS of reversed polarity during which time a visual prey-like stimulus was presented. A negative SPS following positive DC stimulation was associated with enhanced neuronal responses to a visual stimulus. The positive SPS that followed negative stimulation was associated with a decline in neural responses below background when a visual stimulus was additionally given. The SPS was largely a result of DC stimulation that interacted with the motivational tendency to produce enhanced neuronal responses, while the potential was negative and vice versa.