Endo-Porter-mediated delivery of phosphorodiamidate morpholino oligos (PMOs) in erythrocyte suspension cultures from Cope's gray treefrog Hyla chrysoscelis

Cope's gray treefrog, Hyla chrysoscelis, is a freeze-tolerant anuran that accumulates cryoprotective glycerol during cold acclimation. H. chrysoscelis erythrocytes express the aquaglyceroporin HC-3, which facilitates transmembrane glycerol and water movement. Aquaglyceroporins have no pharmacological inhibitors, and no genetic knockout tools currently exist for H. chrysoscelis. A phosphorodiamidate morpholino oligo (PMO)-mediated expression knockdown approach was therefore pursued to provide a model for testing the role of HC-3. We describe a novel procedure optimized for specific, efficient knockdown of HC-3 expression in amphibian erythrocyte suspensions cultured at nonmammalian physiological temperatures using Endo-Porter. Our protocol includes three critical components: pre-incubation at 37°C, two rounds of Endo-Porter and HC-3 PMO administration at ~23°C, and continuous shaking at 190 rpm. This combination of steps resulted in 94% reduction in HC-3 protein expression (Western blot), substantial decrease in HC-3 expression in >65% of erythrocytes, and no detectable expression in an additional 30% of cells (immunocytochemistry).     

Cloning of the 18S rDNA gene, an internal transcribed spacer, and the 5' region of the 28S rDNA gene of Cope's gray treefrog, Hyla chrysoscelis

The location of rDNA genes on the chromosomes of most species is identical within that species, usually occurring on the same chromosome or chromosomes. This is not the case in Cope's gray treefrog, Hyla chrysoscelis, where the rDNA genes are polymorphic for chromosome location. The occasions leading to this polymorphism have yet to be determined. The first step in understanding the nature of the polymorphism is the characterization of the ribosomal gene array. Here we describe the cloning, sequencing, and confirmation, by fluorescence in situ hybridization, of the 18S rDNA gene, a region which includes the end of the 18S rDNA gene, an internal transcribed spacer, and a portion of the 5' end of the 28S rDNA gene in H. chrysoscelis.     

Species specificity and temperature dependency of temporal processing by the auditory midbrain of two species of treefrogs

The mating (advertisement) calls of two sibling species of gray treefrogs, Hyla versicolor and Hyla chrysoscelis, are spectrally identical but differ in trill rate; being higher for H. chrysoscelis. Single-unit recordings were made from the torus semicircularis of both species to investigate the neural mechanisms by which this species-specific temporal feature is analyzed. Using sinusoidally amplitude-modulated (AM) white noise as a stimulus, the temporal selectivity of these midbrain auditory neurons could be described by five response categories: 'AM nonselective' (34%); 'AM high-pass' (7%); 'AM low-pass' (6%); 'AM band-suppression' (12%); 'AM tuned' (40%). The distributions of temporal tuning values (i.e., modulation rate at which each AM-tuned unit responds maximally) are broad; in both species, neurons were found which were tuned to modulation rates greater than those found in their advertisement calls. Nevertheless, the temporal tuning values for H. versicolor (median = 25 Hz) were significantly lower than those for H. chrysoscelis (median = 32.5 Hz). The temporal selectivities of AM band-suppression neurons were found to be temperature dependent. The modulation rate at which a response minimum was observed shifted to higher values as the temperature was elevated. These results extend our earlier findings of temperature-dependent temporal selectivity in the gray treefrog. The selectivity of band-suppression and AM-tuned neurons to various rates of amplitude modulation was largely, but not completely, independent of whether sinusoidal or natural forms of AM were used.     

Field and laboratory studies of the susceptibility of the green treefrog (Hyla cinerea) to Batrachochytrium dendrobatidis infection

Amphibians worldwide are experiencing devastating declines, some of which are due to the amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd). Populations in the southeastern United States, however, have not been noticeably affected by the pathogen. The green treefrog (Hyla cinerea) is abundant and widespread in the southeastern United States, but has not been documented to harbor Bd infection. This study examined the susceptibility of H. cinerea to two strains of Bd in the lab and the prevalence of infection in wild populations of this species in southeastern Louisiana. Although we were able to infect H. cinerea with Bd in the lab, we did not observe any clinical signs of chytridiomycosis. Furthermore, infection by Bd does not appear to negatively affect body condition or growth rate of post-metamorphic individuals. We found no evidence of infection in surveys of wild H. cinerea. Our results suggest that H. cinerea is not susceptible to chytridiomycosis post-metamorphosis and probably is not an important carrier of the fungal pathogen Bd in the southeastern United States, although susceptibility at the larval stage remains unknown.     

Asymmetric competition between Rana clamitans and Hyla versicolor tadpoles

When two organisms interact over a potentially limiting resource, several outcomes are possible (e.g. neither affected, both equally affected, one affected more than the other). The outcome depends on a variety of factors, both internal and external to the organism. We performed a laboratory experiment to investigate the outcome of potential competitive interactions between tadpoles of the green frog ( Rana clamitans ) and the gray treefrog ( Hyla versicolor ), and to examine the factors that may explain the outcome of the interaction. We found that competition between these two species was asymmetric. Hyla versicolor tadpoles were more affected by intraspecific competition than interspecific competition (i.e. grew faster when grown with heterospecifics than conspecifics); whereas, R . clamitans tadpoles were more affected by interspecific competition than intraspecific competition (i.e. grew faster when grown with conspecifics than heterospecifics). Based on behavioral observations, this result could not be explained on the basis of activity levels, a trait previously linked to competitive ability in tadpoles. However, differences in initial body size may help explain the observed results; the gray treefrog tadpoles were larger than the green frog tadpoles.     

Dynamic regulation of aquaglyceroporin expression in erythrocyte cultures from cold- and warm-acclimated Cope's gray treefrog, Hyla chrysoscelis

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.     

Advertisement-call preferences in diploid-tetraploid treefrogs (Hyla chrysoscelis and Hyla versicolor): implications for mate choice and the evolution of communication systems

Signals used for mate choice and receiver preferences are often assumed to coevolve in a lock-step fashion. However, sender-receiver coevolution can also be nonparallel: even if species differences in signals are mainly quantitative, females of some closely related species have qualitatively different preferences and underlying mechanisms. Two-alternative playback experiments using synthetic calls that differed in fine-scale temporal properties identified the receiver criteria in females of the treefrog Hyla chrysoscelis for comparison with female criteria in a cryptic tetraploid species (H. versicolor); detailed preference functions were also generated for both species based on natural patterns of variation in temporal properties. The species were similar in three respects: (1) pulses of constant frequency were as attractive as the frequency-modulated pulses typical of conspecific calls; (2) changes in preferences with temperature paralleled temperature-dependent changes in male calls; and (3) preference functions were unimodal, with weakly defined peaks estimated at values slightly higher than the estimated means in conspecific calls. There were also species differences: (1) preference function slopes were steeper in H. chrysoscelis than in H. versicolor; (2) preferences were more intensity independent in H. chrysoscelis than in H. versicolor; (3) a synergistic effect of differences in pulse rate and shape on preference strength occurred in H. versicolor but not in H. chrysoscelis; and (4) a preference for the pulse shape typical of conspecific calls was expressed at the species-typical pulse duration in H. versicolor but not in H. chrysoscelis. However, females of H. chrysoscelis did express a preference based on pulse shape when tested with longer-than-average pulses, suggesting a hypothesis that could account for some examples of nonparallel coevolution. Namely, preferences can be hidden or revealed depending on the direction of quantitative change in a signal property relative to the threshold for resolving differences in that property. The results of the experiments reported here also predict patterns of mate choice within and between contemporary populations. First, intraspecific mate choice in both species is expected to be strongly influenced by variation in temperature among calling males. Second, simultaneous differences in pulse rate and pulse shape are required for effective species discrimination by females of H. versicolor but not by females of H. chrysoscelis. Third, there is greater potential for sexual selection within populations and for discrimination against calls produced by males in other geographically remote populations in H. chrysoscelis than in H. versicolor.     

Climatic variation and the distribution of an amphibian polyploid complex

The establishment of polyploid populations involves the persistence and growth of the polyploid in the presence of the progenitor species. Although there have been a number of animal polyploid species documented, relatively few inquiries have been made into the large-scale mechanisms of polyploid establishment in animal groups. Herein we investigate the influence of regional climatic conditions on the distributional patterns of a diploid-tetraploid species pair of gray treefrogs, Hyla chrysoscelis and H. versicolor (Anura: Hylidae) in the mid-Atlantic region of eastern North America. Calling surveys at breeding sites were used to document the distribution of each species. Twelve climatic models and one elevation model were generated to predict climatic and elevation values for gray treefrog breeding sites. A canonical analysis of discriminants was used to describe relationships between climatic variables, elevation and the distribution of H. chrysoscelis and H. versicolor. There was a strong correlation between several climatic variables, elevation and the distribution of the gray treefrog complex. Specifically, the tetraploid species almost exclusively occupied areas of higher elevation, where climatic conditions were relatively severe (colder, drier, greater annual variation). In contrast, the diploid species was restricted to lower elevations, where climatic conditions were warmer, wetter and exhibited less annual variation. Clusters of syntopic sites were associated with areas of high variation in annual temperature and precipitation during the breeding season. Our data suggest that large-scale climatic conditions have played a role in the establishment of the polyploid H. versicolor in at least some portions of its range. The occurrence of the polyploid and absence of the progenitor in colder, drier and more varied environments suggests the polyploid may possess a tolerance of severe environmental conditions that is not possessed by the diploid progenitor. Our findings support the hypothesis that increased tolerance to severe environmental conditions is a plausible mechanism of polyploid establishment.     

Comparing bivariate reaction norms among species: time and size at metamorphosis in three species of Hyla (Anura: Hylidae)

Summary Univariate and bivariate methods for comparing norms of reaction among species are discussed and illustrated with an example using North American hylid treefrogs. Norms of reaction for size at metamorphosis (SM) and length of larval period (LP) were compared among treefrog species raised at different food levels (Hyla cinerea vs H. gratiosa) and at different temperatures (H. cinerea vs H. gratiosa vs H. squirella). Hyla cinerea and Hyla gratiosa show parallel norms of reaction across food levels and temperatures. Across temperatures, H. squirella shows a much smaller change in SM relative to change in LP than do H. cinerea and H. gratiosa. This difference in shape of reaction norms may reflect different histories of selection resulting from these species' use of different larval habitats.     

Replication banding patterns of the diploid-tetraploid treefrogs Hyla chrysoscelis and H. versicolor

Populations of the diploid-tetraploid treefrogs Hyla chrysoscelis and H. versicolor can be defined by the polymorphic positions of the nucleolar organizing regions (NORs) on their chromosomes. Evidence from NOR positions and interstitial telomere sequence data shows that gene flow between H. chrysoscelis populations appears to be restricted, with contact occurring only in narrow "hybrid" zones. Hyla versicolor appears to have had multiple origins from H. chrysoscelis populations, and this, too, is reflected in the NOR positions. We used replication banding to determine if genetic isolation of H. chrysoscelis populations was accompanied by karyotype evolution in the populations or in contact zones. We also sought to detect karyotype alteration or replication differences associated with polyploidy in H. versicolor. Homologous chromosome pairs of all H. chrysoscelis studied displayed no differences in replication banding patterns, nor did they differ from those of H. versicolor. Although NOR positions differed between the populations studied, no disturbance of the replication banding patterns was found, indicating that structural rearrangements were not involved in creating the multiple NOR positions seen in populations of H. versicolor and H. chrysoscelis.     

Selectivity for harmonic structure in complex sounds by the green treefrog (Hyla cinerea)

1. A psychophysical technique based on reflex modification was used to study the detection of two-tone complexes in background noise by the green treefrog (Hyla cinerea). Three different two-tone complexes were synthesized and presented to measure detection thresholds--a harmonic complex of 900 + 3000 Hz (periodicity of 300 Hz, mimicking the structure of the natural advertisement call); an inharmonic complex of 830 + 3100 Hz; and a second harmonic complex of 828 + 2760 Hz (periodicity of 276 Hz). 2. Masked thresholds and 'critical ratios' (signal-to-noise ratios at threshold) were lowest for the two harmonic complexes (900 + 3000 Hz, mean 'critical ratio' of 16 dB; 828 + 2760 Hz, mean 'critical ratio' of 14 dB). For the inharmonic complex, for which there is no stable first-harmonic periodicity, the mean 'critical ratio' was 24 dB. These data suggest that the green treefrog is sensitive to the harmonic structure of complex sounds as a specific acoustic feature. 3. Because of the unique structure of the treefrog's inner ear, the heightened behavioral sensitivity to harmonic complexes must be due to processing in the central, rather than peripheral, auditory system.     

Comparative cytochemical measurements in the diploid-tetraploid species pair of hylid frogs Hyla chrysoscelis and H. versicolor.

Hyla versicolor (2n = 48) is a tetraploid counterpart to H. chrysoscelis (2n = 24). Cytochemical measurements revealed that no cytological parameter of the two species conformed to the expected 2:1 ratio. Every cytological factor of the tetraploid appeared to have been regulated toward the diploid level. H. veriscolor-to-H. chrysoscelis mean ratios and their standard errors were: DNA 1.90 +/- 0.03; nuclear histone, 1.70 +/- 0.18; nuclear RNA, 1.63 +/- 0.19; total nuclear protein, 1.46 +/- 0.05; nuclear sizes, 1.42 +/- 0.08.     

Diploid and tetraploid grey treefrogs (Hyla chrysoscelis and Hyla versicolor) have similar metabolic rates

We measured oxygen consumption of the diploid frog Hyla chrysoscelis and its recently evolved tetraploid cryptic species Hyla versicolor at rest and during forced locomotory exercise. We also measured whole-body lactic acid concentrations of individuals of the two species at rest and following 4 min of exercise. Contrary to predictions based on tissue-culture experiments, rates of organismal metabolism are not different in this diploid/tetraploid species pair: the diploid and tetraploid species did not differ in rates of oxygen consumption at rest or during exercise. Furthermore, whole-body lactate concentrations of frogs at rest and following forced locomotion were the same in the diploid and tetraploid species. For these species, polyploidy does not appear to be associated with lower maintenance costs at the level of the organism. However, polyploidy also does not appear to have metabolic consequences that would limit the behavioral or ecological repertoire of an anuran.     

Reproductive character displacement in the acoustic communication system of green tree frogs (Hyla cinerea)

Abstract Interactions between species can affect the evolution of their sexual signals, receiver selectivity, or both. One commonly expected outcome is reproductive character displacement, whereby adverse consequences of mismating select for greater differentiation of communication systems in areas of sympatry than in areas of allopatry. We found evidence of reproductive character displacement in the acoustic communication system of green tree frogs ( Hyla cinerea ). The strength of female preferences for the spectral properties of calls that distinguish conspecific calls from those of a closely related congener, H. gratiosa , was greater in areas of sympatry with H. gratiosa than in areas of allopatry. We also found subtle differences in advertisement calls and in the heights of male calling perches when we restricted our comparisons to localities in which H. gratiosa was also breeding (syntopy) with localities where this species was absent. Hyla cinerea and H. gratiosa show only weak genetic incompatibility, but the calls representative of interspecific hybrids were unattractive to females of both parental species. Hybrids might also be at an ecological disadvantage because of different habitat preferences of the two taxa. Thus, selection against production of less fit or less attractive hybrid or backcross offspring are probably the main causes responsible for the differences documented in this paper.     

Functional equivalence of non-lethal effects: generalized fish avoidance determines distribution of gray treefrog, Hyla chrysoscelis, larvae

How patterns of prey abundance are generated determines how predation influences population and community level dynamics. We investigated how a natural population of ovipositing treefrogs, Hyla chrysoscelis , partitioned their eggs amongst experimental ponds differing in the non-lethal presence of five fish predators, each representing a distinct family. Four fish predators were functionally equivalent in terms of the behavioral response they induced in H. chrysoscelis females and hence the distribution of larval H. chrysoscelis . Mean number of H. chrysoscelis eggs deposited was significantly decreased by the mere presence of these predators relative to controls, with three species eliciting a complete avoidance (e.g. no eggs deposited). One fish predator, Aphredoderus sayanus , was statistically indistinguishable from the control treatment. These data mimic patterns of species distribution observed in nature in response to the presence of fish, but suggest an alternative mechanism for generating patterns of prey abundance amongst habitats differing in their predator composition. Furthermore, our results expand the importance of predator induced non-lethal effects as a process that could dramatically affect population and community level dynamics.     

Ribosomal RNA structure in the diploid and phylogenetically polyploid amphibian species Hyla and Odontophrynus

Ribosomal RNA of the diploid amphibian species Hyla chrysoscelis and Odontophrynus americanus is structurally modified by hidden breaks. Phylogenetically polyploid related species like the tetraploid Hyla versicolor, the tetraploid Odontophrynus americanus and the octoploid Ceratophrys ornata do not show hidden breaks in ribosomal RNA. Structural modifications of rRNA molecules in diploid amphibians has no detectable effect on the ribosomal activity in vitro.     

Replication banding and FISH analysis reveal the origin of the Hyla femoralis karyotype and XY/XX sex chromosomes

The pine woods treefrog, Hyla femoralis, is unique among North American hylid frogs in having a metacentric chromosome 6 and heteromorphic sex chromosomes of the XY/XX type. The X chromosome is distinguished by having a nucleolar organizing region (NOR) in the short arm. The Y chromosome does not possess an NOR. Until the present study, it was not known if the NOR was not present on the Y chromosome or inactive and therefore not detectable by conventional cytogenetic methods like silver staining. Exclusive of its unique features the karyotype of H. femoralis closely resembles those of North American frogs with karyotypes like H. chrysoscelis. We used replication banding and fluorescence in situ hybridization (FISH) with a DNA probe to the 18S + 28S ribosomal genes, which are located at the NOR, to characterize the H. femoralis karyotype. Our analysis revealed that the 18S + 28S ribosomal genes are not present on the Y chromosome, and that the karyotype of H. femoralis was derived from an H. chrysoscelis-like karyotype by relocation of the NOR to the X chromosome from chromosome 6 and either a concurrent or subsequent pericentric inversion of chromosome 6.     

Isolation and characterization of simple sequence repeat loci in the gray tree frog, Hyla chrysoscelis.

A gray tree frog (Hyla chrysoscelis) genomic library was constructed and characterized with regard to the incidence and complexity of simple sequence repeat (SSR) loci. The partial genomic library, containing approximately 10,000 clones with an average-sized insert of 350 bp, was screened with six SSR repeat oligonucleotides (AC, AG, ACG, AGC, AAC, and AAG). Screening identified 31 unique positive clones containing 41 SSR loci. Sequences of tandemly arrayed dinucleotide repeats were more common (36 of 41) than trinucleotide repeats. Twenty-six loci were identified using the AC dinucleotide probe, while 7 loci were identified using the AG dinucleotide probe. An additional 3 AT dinucleotide loci were serendipitously identified. The AT repeats generally comprised the longest dinucleotide repeat loci. The SSR repeat loci reported here should provide potent markers for identity, parentage, and short-lineage determinations in large-scale experiments using gray tree frogs.     

Freeze tolerance in the gray treefrog: cryoprotectant mobilization and organ dehydration.

Freeze tolerance in the frog Rana sylvatica is supported by nonanticipatory mobilization of cryoprotectant (glucose) and redistribution of organ water. Other freeze-tolerant frogs may manifest these responses but differences exist. For example, the gray treefrog (Hyla versicolor) accumulates mostly glycerol as opposed to glucose. The current study reports additional novel features about cryoprotection in H. versicolor. Frogs were acclimated to low temperature for 12 weeks and frozen for 3 days at -2.4 degrees C. Some frogs were then thawed at 3 degrees C for 4 hr. Calorimetry revealed that frozen frogs had 53.9% +/- 11.1% of their body water in ice, and all frogs recovered following this procedure. Plasma glucose was low prior to the onset of freezing (1.1 +/- 0.9 micromol/ml) and it was 20x higher in postfreeze frogs. Constituting nearly 30% of plasma solute, glycerol was 117.2 +/- 13.6 micromol/ml prior to freezing and it remained equally high in postfreeze frogs. Liver water content was moderately lower in frozen frogs when compared to controls (62.9% +/- 3.7% vs. 68.6% +/- 1.7%), whereas postfreeze frogs excessively hydrated their livers (75.7% +/- 2.1%). Less-pronounced changes were seen in muscle water content. H. versicolor can mobilize its major cryoprotectant, glycerol, in response to extended cold acclimation, which is unique in comparison to other freeze-tolerant frogs, and it experiences only moderate organ dehydration during freezing. This species conforms with other freeze-tolerant frogs, however, by mobilizing glucose as a direct response to tissue freezing.     

Respiratory response to temperature and hypoxia in the zebra mussel Dreissena polymorpha

The effects of temperature acclimation, acute temperature variation and progressive hypoxia on oxygen consumption rates (VO2) were determined for the zebra mussel Dreissena polymorpha. In the first experiment, after acclimation to 5, 15 or 25 degrees C for at least 2 weeks, VO2 was determined at 5 degrees C increments from 5 to 45 degrees C. VO2 increased in all three acclimation groups from 5 to 30 degrees C, corresponding to the normal ambient temperature range for this species. Mussels displayed imperfect temperature compensation at temperatures above 15 degrees C, but exhibited little acclimatory ability below 15 degrees C. In the hypoxia experiment, VO2 was determined over the course of progressive hypoxia, from full saturation (oxygen tension [PO2]=160 Torr [21.3 kPa]) to a PO2 at which oxygen uptake ceased (<10 Torr [1.3 kPa]). Mussels were acclimated to either 5, 15 or 25 degrees C for at least 2 weeks and their respiratory response to progressive hypoxia was measured at three test temperatures (5, 15 and 25 degrees C). The degree of oxygen regulation increased with increasing test temperature, particularly from 5 to 15 degrees C, but decreased with increasing acclimation temperature. The decreased metabolic rate observed for warm-acclimated animals, particularly in the upper portion of the temperature range of the zebra mussel, may allow for conservation of organic energy stores during warm summer months. Compared to other freshwater bivalves, D. polymorpha is a relatively poor oxygen regulator, corresponding with its preference for well-oxygenated aquatic habitats. In addition, a new quantitative method for determining the degree of oxygen regulation is presented.