Experimentally induced sexual behavior in male gray treefrogs activates the HPG but not the HPI axis.

At the right time of year across the eastern USA, from Texas to Maine, wetlands come alive with the sound of trilling eastern gray treefrogs (Hyla versicolor), each hoping to attract the attention of a nearby female. However, male eastern gray treefrogs are not the only vociferous suitors in the neighbourhood; they share their wetland breeding grounds with another close relative, Cope's gray treefrogs (Hyla chrysoscelis). ‘The two species are visually indistinguishable’, says Saumya Gupta from the University of Minnesota, USA. Yet, the consequences of a Cope's gray treefrog inadvertently selecting an eastern gray treefrog partner would be disastrous; their offspring rarely survive and, if they do, they are infertile. Fortunately, eastern and Cope's gray treefrogs barely make this rookie mistake, thanks to their trilling croaks. Even though the trill structures of their amorous calls are similar, they are sufficiently different – eastern gray treefrogs trill a little more slowly than their Cope's cousins – for females to avoid selecting the wrong mate. Yet, no one was sure exactly how these closely related species distinguish between each other's trills. However, Gupta and Mark Bee (University of Minnesota) had a hunch. They knew that a unique set of neurons in frog brains – called interval-counting neurons – are often tuned to the trill rate of their own species’ calls. Could these neurons hold the key to female eastern gray treefrogs rebuffing Cope's gray treefrog serenades and vice versa?Collecting female gray treefrogs in amorous embraces with their male partners from the Tamarack Nature Center in Minnesota, USA, Gupta first had to identify which species each frog belonged to. ‘I played mating calls of the eastern and Cope's gray treefrogs from two separate loudspeakers and identified the species based on which speaker it approached’, she says. Next, Gupta simulated a series of gray treefrog trills composed of individual pulses – 10 ms pulses, 20 ms apart for the Cope's gray treefrogs and 30 ms pulses separated by 60 ms for the eastern gray treefrogs. After recreating the distinctive pulse signatures of both species, she played a range of trills to the females, from a single pulse up to 16 repetitions, recording the number of pulses when the female's attention switched from indifference to attraction as she hopped toward the loudspeaker.Comparing the females’ reactions, it was clear that the Cope's gray treefrogs tended to become interested in trills composed of ∼8, 10 ms pulses when the calls were softer (65 dB SPL) while trills of ∼7.5 pulses became attractive when louder (85 dB SPL). In contrast, the eastern gray treefrog females only required ∼5, 30 ms pulses to get their attention, falling to 3.5 pulses when the volume was higher. But how would the interval-counting neurons in the treefrog brain respond to each species-specific serenade?This time, Rishi Alluri and Gary Rose from the University of Utah, USA, played simulated trills with increasing numbers of pulses to the frogs while painstakingly recording when interval-counting neurons in the frog brain began sending electrical nerve signals. Eventually, the duo identified a subset of neurons in the Cope's gray treefrog brain tuned to fire when they heard trills of at least 6–8, 10 ms pulses. Meanwhile, the eastern gray treefrogs’ interval-counting neurons responded strongly when the frogs heard trills containing at least 3–5, 30 ms pulses.‘Interval-counting neurons play key roles in decoding information about species identity in frogs’, says Gupta, placing the neurons at the hub of the mechanism protecting eastern and Cope's gray treefrogs from interbreeding, keeping them faithful to their own species’ serenades.

This study compares the helminth faunas between Cope's gray treefrogs ( Hyla chrysoscelis ) and green treefrogs ( Hyla cinerea ), in areas where they have recently overlapped due to range expansion by H. cinerea , to determine whether or not 2 species of frogs with a high degree of similarity in many of their life history traits also exhibit similarities in the composition of their helminth assemblages. Results of this study did not find significant differences in helminth species diversity when sympatric and allopatric populations of the same species of frog were compared. There was, however, a significant difference in helminth diversity among sympatric populations of H. chrysoscelis and H. cinerea , and this difference was in large part attributable to the significantly higher abundance of the gastrointestinal nematode Cosmocercoides variabilis among H. chrysoscelis . Additional studies will be required to determine whether the observed patterns are due to differences in arrival time, perch locations within the chorus, or parasite-mediated competition.

This study tested three hypotheses about the ability of female frogs to exploit temporal fluctuations in the level of background noise to overcome the problem of recognizing male advertisement calls in noisy breeding choruses. Phonotaxis tests with green treefrogs (Hyla cinerea) and Cope's gray treefrogs (Hyla chrysoscelis) were used to measure thresholds for recognizing calls in the presence of noise maskers with (a) no level fluctuations, (b) random fluctuations, or level fluctuations characteristic of (c) conspecific choruses and (d) heterospecific choruses. The dip-listening hypothesis predicted lower signal recognition thresholds in the presence of fluctuating maskers compared with nonfluctuating maskers. Support for the dip-listening hypothesis was weak; only Cope's gray treefrogs experienced dip listening and only in the presence of randomly fluctuating maskers. The natural soundscapes advantage hypothesis predicted lower recognition thresholds when level fluctuations resembled those of natural soundscapes compared with artificial fluctuations. This hypothesis was rejected. In noise backgrounds with natural fluctuations, the species-specific advantage hypothesis predicted lower recognition thresholds when fluctuations resembled species-specific patterns of conspecific soundscapes. No evidence was found to support this hypothesis. These results corroborate previous findings showing that Cope's gray treefrogs, but not green treefrogs, experience dip listening under some noise conditions. Together, the results suggest level fluctuations in the soundscape of natural breeding choruses may present few dip-listening opportunities. The findings of this study provide little support for the hypothesis that receivers are adapted to exploit level fluctuations of natural soundscapes in recognizing communication signals.

In your CORT: Corticosterone and its receptors in the brain underlie mate choosiness in female Cope's gray treefrogs (Hyla chrysoscelis)

Female frogs discriminate among potential mates based on individual variation in male advertisement calls. While considerable data have accumulated allowing comparisons of female preference functions among species, we still lack fundamental knowledge about how and why the shapes of preference functions for particular call properties vary among populations within all but a few species. Here, we report results from a study aimed at describing female preference functions for spectral call properties in Cope's gray treefrog (Hyla chrysoscelis). Widespread throughout the eastern half of North America, Cope's gray treefrog is the diploid member of the cryptic diploid-tetraploid Hyla versicolor species complex, and its populations are divided into two distinct genetic lineages (eastern and western). In this study of a western lineage population, we recorded and analyzed the spectral properties of 1000 advertisement calls from 50 males and conducted two-choice phonotaxis experiments to estimate a population-level preference function. Females preferred calls with average frequencies over calls with frequencies that were 2 or 3 semitones (1.4 or 2.1 standard deviations, respectively) lower than the population mean. We observed no behavioral discrimination between calls with average and higher-than-average frequencies. Preferences discriminating against low-frequency calls were weak and were abolished by attenuating the preferred average call by 3 dB. We discuss these results in light of previous studies of eastern lineage populations, geographic variation in female preference functions, and the potential adaptive value of discriminating against calls with low frequencies.

The difference a day makes: Breeding remodels hearing, hormones and behavior in female Cope's gray treefrogs (Hyla chrysoscelis)

Cope's Gray Tree Frog, Hyla chrysoscelis is a freeze tolerant anuran that accumulates glycerol during cold acclimation. We hypothesize that the osmoregulatory capabilities of erythrocytes‐‐ to tolerate changes in cell volume and to affect rapid water and solute fluxes‐‐are likely to be most important during the events of freezing and thawing, and are dependent upon the expression and function of HC‐3, an ortholog of the aquaglyceroporin AQP3. Erythrocytes of H. chrysoscelis cultured for 48 hrs in media made hyperosmotic (400 mOsM) through the addition of 150 mM glycerol or urea showed enhanced HC‐3 glycosylation and membrane localization compared to those cultured in control or hypertonic media (250 mOsM culture media + 150 mM sorbitol or 75 mM NaCl). Cultured erythrocytes transferred from hyperosmotic culture media (glycerol or urea) to a hypotonic solution containing 250 mM glycerol or urea underwent a series of shape changes as they swelled, initially elongating, then becoming swollen and round. Cells cultured in hypertonic media (sorbitol or NaCl) responded with significantly less osmotically‐induced swelling (i.e. cell shape change). When aquaporins were blocked by HgCl2, cells retained normal dimensions, indicating the importance of aquaporins in conferring water and solute permeability in these cells. This research was supported by NSF Research Grant IOS‐1121457.

32. Jumping genes in frogs; cytogenetic evidence that additional ribosomal gene locations in Hyla chrysoscelis (Cope's gray treefrog) are associated with mobile genetic elements

Organisms belonging to the genus Staphylococcus were isolated on mannitol salt agar from the feces of wild caught Cope's gray treefrogs (Hyla chrysoscelis) from east-central Kansas. All 222 presumptive isolates were confirmed as coagulase-negative staphylococci with Staphylococcus sciuri and Staphylococcus xylosus being most prevalent. Antibiotic susceptibility patterns to five different antibiotics were determined and the results indicated 99% of all isolates were resistant to penicillin G and 59% of the isolates were resistant to oxacillin, a clinical substitute for methicillin. Due to the significance of methicillin resistance in the genus Staphylococcus, 10 randomly chosen oxacillin resistant organisms were analyzed for the presence of the mecA gene, which is known to code for methicillin resistance. The gene was detected in four of the 10 organisms examined. These data indicate that gray treefrogs are harboring inordinately large numbers of methicillin resistant staphylococci as part of their normal flora and that the mechanism of methicillin resistance may be independent of mecA.

Urban stormwater runoff is composed of a mixture of components, including polycyclic aromatic hydrocarbons, metals, deicing agents, and many others. The fate of these chemicals is often in stormwater detention ponds that are used by amphibians for breeding. Among aquatic organisms, the toxic mechanism for many metals involves interference with active Na(+) and Cl(-) uptake. Addition of cations has been shown to reduce the toxicity of metals among some aquatic organisms through competitive inhibition, but no studies have investigated the interaction between NaCl and Cu among amphibian embryos and larvae. To determine the degree to which NaCl may ameliorate the toxicity of Cu to amphibian embryos and larvae, the authors exposed Hyla chrysoscelis (Cope's gray treefrogs) and Rana (Lithobates) clamitans (green frogs) to seven levels of Cu and NaCl in fully factorial experiments. When exposure was in artificial hard water, Cu was highly toxic to both species (96-h median lethal concentration [LC50] of 44.7 µg/L and 162.6 µg/L for H. chrysoscelis and R. clamitans, respectively). However, approximately 500 mg/L of NaCl eliminated Cu toxicity over the range of Cu concentrations used in the experiments (maximum 150 µg Cu/L for H. chrysoscelis and 325 µg Cu/L for R. clamitans). The current results suggest that NaCl is likely responsible for the toxic effects of NaCl and metal mixtures that might be typical of runoff from road surfaces in northern latitudes.

Cope's gray tree frog Hyla chrysoscelis accumulates glycerol during cold acclimation. We hypothesized that, during this process, gray tree frogs adjust renal filtration and/or reabsorption rates to retain accumulated glycerol. During cold acclimation, plasma concentrations of glycerol rose >200-fold, to 51 mmol/l. Although fractional water reabsorption decreased, glomerular filtration rate (GFR) and, consequently, urine flow were <5% of warm levels, and fractional glycerol reabsorption increased. In contrast, dehydrated frogs increased fractional water reabsorption, decreased GFR, and did not accumulate glycerol. We hypothesized that expression of proteins from the aquaporin (AQP)/glyceroporin (GLP) family was associated with changing patterns of water and glycerol movement. We cloned the cDNA for three such proteins, quantified mRNA expression in nine tissues using real-time quantitative PCR, and functionally characterized them using a Xenopus oocyte expression system. HC-1, an AQP1-like water channel conferring low glycerol permeability, is expressed ubiquitously in warm- and cold-acclimated tissues. HC-2, a water channel most similar to AQP2, is primarily expressed in organs of osmoregulation. HC-3, which is most similar to AQP3, is functionally characterized as a GLP, with low permeability to water but high permeability to glycerol. Aspects of expression levels and functional characteristics varied between cold and warm conditions for each of the three AQPs, suggesting a complex pattern of involvement in osmoregulation related to thermal acclimation.

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

Ovipositing amphibians select breeding sites that determine their offspring's larval habitat. Preference–performance theory predicts that adults will select habitat patches that match the expected performance of their offspring in those patches. Many amphibians breed in temporary ponds immediately after they fill, and one species, Hyla chrysoscelis (Cope's Gray Treefrogs), selects between breeding sites based on patch age differences of as little as 7 d. Prior work established that H. chrysoscelis have better larval performance in more recently filled ponds, but not in ponds less than 15 d after filling or among ponds that differed in age by less than 65 d. Our objective was to determine experimentally whether larval H. chrysoscelis performance varies across short time scales after filling (3- and 23-d-old ponds) and across two larval densities (15 and 30 larvae per mesocosm). Higher larval densities reduced metamorph mass and body condition, but did not otherwise affect larval performance. There...

Cope's gray treefrog, Hyla chrysoscelis,is a freeze-tolerant anuran which accumulates and distributes glycerol as a cryoprotectant before freezing. We hypothesize that HC-3, an aquaglyceroporin member of the MIP family of water pores, may play an important role in the process of freeze tolerance by mediating transmembrane passage of glycerol and water during cold-acclimation. The objectives of this study were two-fold: to examine HC-3 protein abundance and cellular localization in erythrocytes from cold- and warm-acclimated frogs and to develop and characterize an erythrocyte cell culture system for examining HC-3 gene regulation. Compared with warm-acclimated frogs, erythrocytes from cold-acclimated frogs had higher HC-3 protein expression and enhanced plasma membrane localization. Furthermore, erythrocytes from cold- and warm-acclimated frogs maintained in culture at 4 and 20°C exhibited time- and temperature-dependent regulation of HC-3 expression and an increase in the abundance of high molecular weight immunoreactive species within 24 hr of culture at 20°C. Deglycosylation of erythrocyte proteins resulted in the disappearance of the high molecular weight species, indicating that HC-3 is post-translationally modified by N-linked glycosylation. Erythrocytes cultured in media containing glycerol also showed an increased abundance of the high molecular weight bands and enhanced plasma membrane localization of HC-3, suggesting a role for glycerol in regulating HC-3 subcellular trafficking. Thus, the development of this erythrocyte cell culture system from H. chrysoscelis opened an opportunity to study the properties of cells with changing expression of an aquaglyceroporin, HC-3, and to explore the factors regulating that expression.

In Cope's gray treefrog (Hyla chrysoscelis), thresholds for recognizing conspecific calls are lower in temporally modulated noise backgrounds compared with unmodulated noise. The effect of modulated noise on discrimination among different conspecific calls is unknown. In quiet, females prefer calls with relatively more pulses. This study tested the hypotheses that noise impairs selectivity for longer calls and that processes akin to dip listening in modulated noise can ameliorate this impairment. In two-stimulus choice tests, female subjects were allowed to choose between an average-length call and a shorter or longer alternative. Tests were replicated at two signal levels in quiet and in the presence of chorus-shaped noise that was unmodulated, modulated by a sinusoid, or modulated by envelopes resembling natural choruses. When subjects showed a preference, it was always for the relatively longer call. Noise reduced preferences for longer calls, but the magnitude of this reduction was unrelated to whether the noise envelope was modulated or unmodulated. Together, the results are inconsistent with the hypothesis that dip listening improves a female gray treefrog's ability to select longer calls in modulated compared with unmodulated noise.