Tympanic and extratympanic sound transmission in the leopard frog

Summary The inner ear of the leopard frog,Rana pipiens, receives sound via two separate pathways: the tympanic-columellar pathway and an extratympanic route. The relative efficiency of the two pathways was investigated. Laser interferometry measurements of tympanic vibration induced by free-field acoustic stimulation reveal a broadly tuned response with maximal vibration at 800 and 1500 Hz. Vibrational amplitude falls off rapidly above and below these frequencies so that above 2 kHz and below 300 Hz tympanic vibration is severely reduced. Electrophysiological measurements of the thresholds of single eighth cranial nerve fibers from both the amphibian and basilar papillae in response to pure tones were made in such a way that the relative efficiency of tympanic and extratympanic transmission could be assessed for each fiber. Thresholds for the two routes are very similar up to 1.0 kHz, above which tympanic transmission eventually becomes more efficient by 15–20 dB. By varying the relative phase of the two modes of stimulation, a reduction of the eighth nerve response can be achieved. When considered together, the measurements of tympanic vibration and the measurements of tympanic and extratympanic transmission thresholds suggest that under normal conditions in this species (1) below 300 Hz extratympanic sound transmission is the main source of inner ear stimulation; (2) for most of the basilar papilla frequency range (i.e., above 1.2 kHz) tympanic transmission is more important; and (3) both routes contribute to the stimulation of amphibian papilla fibers tuned between those points. Thus acoustic excitation of the an uran's inner ear depends on a complex interac tion between tympanic and extratympanic sound transmission.Abbreviations dB SPL decibels sound pressure level re: 20 N/ m² - AP amphibian papilla - BP basilar papilla - BEF best excitatory frequency     

Taste responses to amino acids in the southern leopard frog, Rana sphenocephala

Integrated taste recordings of the glossopharyngeal (IX) nerve innervating the tongue of the southern leopard frog were studied in response to various amino acids and quinine hydrochloride. Amino acids and quinine hydrochloride elicited primarily phasic taste responses. Acidic (L-aspartic and L-glutamic) and basic (L-lysine and L-arginine) amino acids, adjusted to pH8, were effective taste stimuli. All glossopharyngeal nerve twigs that responded to amino acid stimuli also responded to quinine; however, not all quinine-sensitive IX nerve bundles were responsive to amino acids. Electrophysiological thresholds for amino acids were estimated to be 2.5-10 mM, whereas threshold for quinine hydrochloride averaged approximately 10 microM.     

Evidence for a major histocompatibility complex in the leopard frog

Using the technique of gynogenetic diploidy, which is uniquely suited to amphibians, populations of frog siblings can be obtained in which the allelic diversity at any given gene locus, including histocompatibility loci, is greatly reduced. Analyses of the survival times of skin allografts exchanged among pairs of gynogenetic diploid larvae indicate that allograft rejection is mediated principally by a major histocompatibility complex that codes for strong H-antigens rather than by the cumulative effects of weak antigens controlled by numerous, independently assorting, loci.     

Contribution of the submentalis muscle to feeding mechanics in the leopard frog, Rana pipiens

This study investigated the functional contributions of the submentalis muscle to the coordination of feeding behavior in the leopard frog, Rana pipiens. Additionally, the anatomical origins of the motor neurons innervating this muscle are identified and described. The m. submentalis is a small muscle connecting the distal mandibular tips. Depending upon the anuran species studied, this muscle contributes to mandibular bending and the degree to which the tongue is protracted, or has little or no role in feeding biomechanics. High-speed videography was used to quantify feeding attempts before versus after bilateral denervation of the m. submentalis. Additionally, the terminal branch of the trigeminal nerve prior to innervating the m. submentalis was retrogradely labeled to identify the origins of motor neurons innervating the muscle. For the kinematic analyses, denervation of the submentalis resulted in significant increases in the time to maximum tongue protrusion, and the duration of tongue protrusion. Neither mandibular bending, nor tongue length variables differed significantly between normal conditions and deafferented conditions. However, when unsuccessful feeding attempts were quantified following the denervation, failed attempts were nearly always due to the tongue not reaching the prey. None of the unsuccessful feedings prior to denervation were due to inadequate tongue protrusion. Anatomical data show a much larger rostral-caudal distribution of the trigeminal motor neurons than previously described for anurans. These data suggest a larger role for the submentalis muscle in Rana than in previously studied anurans with long protrusible tongues, and suggests a feedback mechanism from the trigeminal nerve to the nerves coordinating tongue protraction and retraction.     

Prevalence of Batrachochytrium dendrobatidis in American bullfrog and southern leopard frog larvae from wetlands on the Savannah River Site, South Carolina

Batrachochytrium dendrobatidis, an aquatic fungus, has been linked to recent amphibian population declines. Few surveys have assessed B. dendrobatidis infections in areas where the disease is suggested to be less virulent and population declines have not been observed, such as southeastern North America. Although adult Rana catesbeiana and Rana sphenocephala from the Savannah River Site, South Carolina collected in 1979 and 1982 were identified as having B. dendrobatidis, it is unknown whether the fungus is currently present at the site or if susceptibility to infection varies among species or wetlands with different histories of environmental contamination. From 15 May through 15 August 2004, we collected R. catesbeiana and R. sphenocephala tadpoles from three wetlands with differing contamination histories on the Savannah River Site, South Carolina. We found B. dendrobatidis in only one of the wetlands we surveyed. Batrachochytrium dendrobatidis infection was identified in 64% of the R. catesbeiana tadpoles sampled and histologically assessed (n=50) from a wetland contaminated with mercury, copper, and zinc. No R. sphenocephala tadpoles from this site (n=50) were infected. In combination with a recently published report, our data suggest that B. dendrobatidis has been present at the Savannah River Site for over 25 yr but has not caused any apparent population declines. This time period is similar to the known presence of 30 yr of B. dendrobatidis in northeastern North America. Our data suggest that R. sphenocephala larvae might be resistant to infection, even when occupying the same wetland as the infected R. catesbeiana. Our survey did not clarify the effects of environmental contamination on infection severity, but our study stresses the importance of additional field surveys to document how this pathogen is affecting amphibians globally.     

Vitellogenic cycles in laboratory-maintained females of the leopard frog, Rana pipiens

As a part of studies on the reproduction of laboratory maintained frogs, wild-caught Rana pipiens were ovulated and maintained at 22-27 degrees C for up to 18 months. Vitellogenic oocytes were periodically staged and counted, and a "maturity index" was calculated to assess the progress of the vitellogenic cycle. The initial cycle was similar to that of wild frogs except that the first oocytes to reach stage 5 (mature eggs) usually began to degenerate before later starting oocytes became mature. In addition, a second cycle began before the first was completed. After more than 1 year at room temperature, abnormal cycles were common. Ovaries of such animals contained very few mature eggs. Many of their oocytes were in early stages of vitellogenesis or, if pigmented, had begun to degenerate. These deficiencies were partially corrected in females placed in 4 degrees C for 4-6 weeks. The average number of mature eggs increased 15-fold and ovary weights more than doubled. Oviduct weights almost doubled. Although the rates of cooling, photoperiod, and nutritional status could be important influences, the results imply that cold treatment alone increases estrogen secretion. We suggest that low estrogen secretion may account for the reproductive deficiencies seen in R. pipiens cultured at room temperature.     

Spatial and spectral dependence of the auditory periphery in the northern leopard frog

We investigated directionalities of eardrum vibration and auditory nerve response in anesthetized northern leopard frogs (Rana pipiens pipiens). Simultaneous measures of eardrum velocities and firing rates from 282 auditory nerve fibers were obtained in response to free-field sounds from eight directions in the horizontal plane. Sound pressure at the external surface of the ipsilateral eardrum was kept constant for each presentation direction (± 0.5 dB). Significant effects of sound direction on eardrum velocity were shown in 90% of the cases. Maximum or minimum eardrum velocity was observed more often when sounds were presented from the lateral and posterior fields, or from the anterior and contralateral fields, respectively. Firing rates of 38% of the fibers were significantly affected by sound direction and maximum or minimum firing rate was observed more frequently when sounds were delivered from the lateral fields, or from the anterior and contralateral fields, respectively. Directionality patterns of eardrum velocity and nerve firing also vary with sound frequency. Statistically significant correlation between eardrum velocity and nerve fiber firing rate was demonstrated in only 45% of the fibers, suggesting that sound transmission to the inner ear through extratympanic pathways plays a non-trivial role in the genesis of directionality of auditory nerve responses.Abbreviations CF characteristic frequency - SVL snout-vent length - TM tympanic membrane     

Cytoplasmic microtubules of normal and tumor cells of the leopard frog

Abstract. The cytoplasmic microtubule complex (CMTC) was examined in monolayer cultures of normal tadpole mesonephros, primary renal adenocarcinoma, and an established cell line derived from a pronephric renal adenocarcinoma (PNKT-4B) of the leopard frog, Rana pipiens. Immunocytochemistry revealed typical arrays of microtubules extending from the cytocentrum to the cell periphery in all three cell types when cultured at 28° C; similar results were obtained at 20° C. However, the CMTC was disorganized in both tumor types, in contrast to the retention of a typical CMTC in normal tissue cultured at 7° C. The response of PNKT-4B cells differed from that of normal tadpole mesonephros when treated with the microtubule inhibitor drug nocodazole. At 28° C, PNKT-4B and tadpole mesonephros cells lost their CMTC with nocodazole treatment, and both were able to reconstitute CMTC when nocodazole was removed. Similarly, both lost CMTC organization with nocodazole and culture at 7° C. However, while normal cells could effect a recovery at 7° C after the removal of nocodazole, PNKT4B cells were unable to restructure CMTC under the same conditions. Metastasis in the frog renal adenocarcinoma is temperature-dependent, with an elevated prevalence of metastasis in tumor-bearing frogs maintained at 28° C. Few metastatic colonies are detected in tumor-bearing frogs maintained at a low temperature (7° C). Other studies have indicated that microtubules, which are essential for cell motility, play an important role in the invasion by tumor cells of normal tissue fragments in vitro. The effects of temperature on metastasis of the Lucke renal adenocarcinoma are consistent with temperature-mediated changes in tumor-cell CMTC.     

Histogenesis of the lymphomyeloid complex in the larval leopard frog, Rana pipiens

A histological study was undertaken of the differentiation of the lymphomyeloid complex of larvae of the common leopard frog, Rana pipiens, reared at 18–21 °C. Immature lymphocytes are first recognised in the thymus and pronephros at stage I of Taylor and Kollros ('46). By the end of stage II, small lymphocytes are regularly found among the predominant larger lymphoid cells in the thymus, in which corticomedullary differentiation has begun. At this time, a few small lymphocytes are also apparent in the ventral cavity bodies, lymph gland, pronephros, mesonephros and intestine, but rarely occur in the spleen. During stages III and IV extensive development of these components of the lymphomyeloid complex occurs. The organs now contain large numbers of mature lymphocytes and have attained states of differentiation that remain essentially similar in subsequent larval stages. By stage V, small epithelium-associated lymphoid accumulations are abundant throughout the length of the gastrointestinal tract. The anlagen of the procoracoid body and bone marrow appear just prior to metamorphosis. The kidneys are the main sites of blood formation in the larva. Masses of granulocytes are also usually found in the abundant ventral cavity bodies. A consideration of the roles of each of the organs provides insight into the ontogeny of the immune system of the Anura in general.     

An analysis of selection on a colour polymorphism in the northern leopard frog

In this study, we investigated the role of selection in the maintenance of a dorsal colour polymorphism in natural populations of the northern leopard frog, Rana pipiens. We determined genetic structure both spatially and temporally from a suite of putatively neutral molecular markers and tested whether or not the colour locus exhibited patterns of genetic variation that differed from those of the neutral loci. Spatial genetic structure at the colour locus was indistinguishable from structure at neutral loci [95% confidence intervals of F(ST) (neutral) = (0.07, 0.35), F(ST) (colour locus) = 0.114]. In the temporal analysis, we found that the variance among populations in the change in allele frequency at the colour locus (equal to 0.004) lies within the 95% confidence intervals for the variance among populations in changes in allele frequencies at neutral loci. In light of our inability to show evidence for the selective maintenance of the colour polymorphism, we used computer simulations to infer the power of our spatial and temporal techniques to detect selection. The computer simulations showed that although the strength of selection (s) would need to be relatively strong to have been detected by the temporal approach (s = 0.1-0.4, depending on the model tested), the spatial analysis would have detected all but weak selection (s = 0.01-0.04, depending on the model tested). This study illustrates the importance of using a locus comparison approach to detect evidence for selective maintenance before conducting studies to measure the selective mechanisms maintaining a polymorphism.     

Observations on the development of unusual melanization of leopard frog ventral skin

The ontogeny of ventral pigmentation of two species of leopard frog, Rana pipiens and R. chiricahuensis, was examined by light microscopy and transmission electron microscopy to reveal how the unusual melanistic ventral pigmentation of R. chiricahuensis is achieved at the cellular level. Ventral skin of R. pipiens is always white. Ventral skin of adult R. chiricahuensis is white when frogs are background-adapted to a white substrate, but ventral skin becomes nearly as dark colored as the dorsal skin when frogs darken in response to a black background. Skin samples from tadpoles of both species, newly metamorphosed frogs, and adult frogs were analyzed for chromatophore composition and distribution. Ventral skin of R. pipiens larvae, newly metamorphosed frogs, and adults and of R. chiricahuensis larvae was white due to abundant iridophores and no melanophores. Melanophore density in the ventral integument of R. chiricahuensis was 9.1 ± 2.8/mm² in newly metamorphosed frogs and 87.0 ± 4.8/mm² in adult frogs. Pigment within ventral melanophores migrated during physiological color change during background adaptation. © 1993 Wiley-Liss, Inc.