Sexual dimorphism in external morphology of the American bullfrog Rana (Aquarana) catesbeiana and the possibility of sex determination based on tympanic membrane/eye size ratio.

The American bullfrog Rana (Aquarana) catesbeiana has been reported to show significant sexual dimorphism based on the size ratio between the tympanic membrane and the eye. In males the tympanic membrane is much larger than the eye, but not in females. The ratio has been used as a convenient criterion to discriminate sexes (sexing) in the American bullfrog, though its reliability is unknown. In this study, we examined 86 adult American bullfrogs to clarify whether the tympanic membrane long diameter/eye long diameter (Dtm/De) ratio is a reliable index to discriminate sexes in this species. In addition, we examined the growth of this sexually dimorphic trait. Results indicated that there is a significant difference but there is a small overlap in this ratio Dtm/De between sexes. The allometric comparisons showed the sexual dimorphism of the Dtm/De ratio was increased during growth and the dimorphism is attributable to the difference in the growth rate of the tympanic membrane (Dtm). Therefore, sex determination of American bullfrogs cannot be wholly reliably achieved by the Dtm/De ratio alone; other external morphological features are required in addition.

Histology of the Urogenital System in the American Bullfrog (Rana catesbeiana), with Emphasis on Male Reproductive Morphology.

Previous studies have revealed variations in the urogenital system morphology of amphibians. Recently, the urogenital system of salamanders was reviewed and terminology was synonymized across taxa. Discrepancies exist in the terminology describing the urogenital system of anurans, which prompted our group to develop a complete, detailed description of the urogenital system in an anuran species and provide nomenclature that is synonymous with those of other amphibian taxa. In Rana catesbeiana, sperm mature within spermatocysts of the seminiferous tubule epithelia and are transported to a series of intratesticular ducts that exit the testes and merge to form vasa efferentia. Vasa efferentia converge into single longitudinal ducts (Bidder's ducts) on the lateral aspects of the kidneys. Branches from the longitudinal ducts merge with genital kidney renal tubules through renal corpuscles. The nephrons travel caudally and empty into the Wöffian ducts. Similar to salamanders, the caudal portion of the kidneys (termed the pelvic kidneys in salamanders) only possesses nephrons involved in urine formation, not sperm transport. Data from the present study provide a detailed description and synonymous nomenclature that can be used to make future comparative analyses between taxa more efficient.

Fluoromicrometry: A Method for Measuring Muscle Length Dynamics with Biplanar Videofluoroscopy.

Accurate measurements of muscle length changes are essential for understanding the biomechanics of musculoskeletal systems, and can provide insights into muscular work, force, and power. Muscle length has typically been measured in vivo using sonomicrometry, a method that measures distances by sending and receiving sound pulses between piezoelectric crystals. Here, we evaluate an alternative method, fluoromicrometry, which measures muscle length changes over time by tracking the three-dimensional positions of implanted, radio-opaque markers via biplanar videofluoroscopy. To determine the accuracy and precision of fluoromicrometry, we simultaneously measured length changes of an isolated muscle, the frog sartorius, in an in vitro setup using both fluoromicrometry and a servomotor. For fluoromicrometry to perfectly match the results of the servomotor, the relationship between the two measurements should be linear, with a slope of 1. Measurements of muscle shortening from fluoromicrometry and the motor were compared across 11 isotonic contractions. The precision of fluoromicrometry was ±0.09 mm, measured as the root mean square error of the regression of fluoromicrometry versus servomotor muscle lengths. Fluoromicrometry was also accurate: the mean slope of the fluoromicrometry-servomotor regressions did not differ significantly from the ideal line once off-axis motion was removed. Thus, fluoromicrometry provides a useful alternative for measuring muscle length, especially in studies of live animals, as it permits long-term marker implantation, wireless data collection, and increased spatial sampling. Fluoromicrometry can also be used with X-Ray Reconstruction of Moving Morphology to simultaneously measure muscle shortening and skeletal kinematics, providing a potent new tool for biomechanics research.

Effects of glyphosate and the glyphosate based herbicides Roundup Original(®) and Roundup Transorb(®) on respiratory morphophysiology of bullfrog tadpoles.

Glyphosate-based herbicides are widely used in agriculture and are commonly found in water bodies. Roundup Original(®) (RO) contains an isopropylamine glyphosate (GLY) salt containing the surfactant POEA, while Roundup Transorb R(®) (RTR) contains a potassium salt of GLY with unknown surfactants. Both contain different compositions of so-called "inert" ingredients, more toxic than glyphosate. Amphibian tadpoles often experience variations in O2 availability in their aquatic habitats; an ability to tolerate hypoxia can condition their survival and fitness. We evaluated the impacts of sublethal concentrations of GLY (1 mg L(-1)), RO (1 mg L(-1) GLY a.e) and RTR (1 mg L(-1) GLY a.e) on metabolic rate (V·O2 - mLO2 Kg1 h(-1)) of bullfrog tadpoles during normoxia and graded hypoxia, and related this to morphology of their skin, their major site of gas exchange. In control (CT) V·O2 remained unaltered from normoxia until 40 mmHg, indicating a critical O2 tension between 40 and 20 mmHg. GLY significantly reduced V·O2, possibly due to epidermal hypertrophy, which increased O2 diffusion distance to O2 uptake. In contrast, RTR increased V·O2 during hypoxia, indicating an influence of "inert" compounds and surfactants. V·O2 of RO did not differ from CT, suggesting that any increase in V·O2 caused by exposure was antagonized by epidermal hypertrophy. Indeed, all herbicides caused marked alterations in skin morphology, with cell and epithelium wall presenting hyperplasia or hypertrophy and chromatid rupture. In summary, GLY, RO and RTR exert different effects in bullfrog tadpoles, in particular the surfactants and inert compounds appear to influence oxygen uptake.

Structure and composition of arytenoid cartilage of the bullfrog (Lithobates catesbeianus) during maturation and aging.

The aging process induces progressive and irreversible changes in the structural and functional organization of animals. The objective of this study was to evaluate the effects of aging on the structure and composition of the extracellular matrix of the arytenoid cartilage found in the larynx of male bullfrogs (Lithobates catesbeianus) kept in captivity for commercial purposes. Animals at 7, 180 and 1080 days post-metamorphosis (n=10/age) were euthanized and the cartilage was removed and processed for structural and biochemical analysis. For the structural analyses, cartilage sections were stained with picrosirius, toluidine blue, Weigert's resorcin-fuchsin and Von Kossa stain. The sections were also submitted to immunohistochemistry for detection of collagen types I and II. Other samples were processed for the ultrastructural and cytochemical analysis of proteoglycans. Histological sections were used to chondrocyte count. The number of positive stainings for proteoglycans was quantified by ultrastructural analysis. For quantification and analysis of glycosaminoglycans were used the dimethyl methylene blue and agarose gel electrophoresis methods. The chloramine T method was used for hydroxyproline quantification. At 7 days, basophilia was observed in the pericellular and territorial matrix, which decreased in the latter over the period studied. Collagen fibers were arranged perpendicular to the major axis of the cartilaginous plate and were thicker in older animals. Few calcification areas were observed at the periphery of the cartilage specimens in 1080-day-old animals. Type II collagen was present throughout the stroma at the different ages. Elastic fibers were found in the stroma and perichondrium and increased with age in the two regions. Proteoglycan staining significantly increased from 7 to 180 days and reduced at 1080 days. The amount of total glycosaminoglycans was higher in 180-day-old animals compared to the other ages, with marked presence of chondroitin- and dermatan-sulfate especially in this age. The content of hydroxyproline, which infers the total collagen concentration, was higher in 1080-day-old animals compared to the other ages. The results demonstrated the elastic nature of the arytenoid cartilage of L. catesbeianus and the occurrence of age-related changes in the structural organization and composition of the extracellular matrix. These changes may contribute to alter the function of the larynx in the animal during aging.

Effects of maturation and aging on the pressure-bearing region of the plantaris longus tendon of the bullfrog (Lithobates catesbeianus).

The plantaris longus tendon (PLT) in bullfrog develops a fibrocartilage-like tissue in the area that is functionally subject to compressive forces. The aim of this study was to analyze the modifications of the pressure-bearing region in bullfrog PLT with different ages (7, 180, and 1,080 days after the end of metamorphosis) using histomorphometric, ultrastructural and biochemical methods. Weak basophilia and cells with a fibroblastic phenotype were observed in the compression region at 7 days of age. On the other hand, a large area of intense tissue basophilia associated with a chondroblast-like cell population was noted at the other ages. Collagen fibers exhibited a three-dimensional network-like arrangement at all ages. The number of connective tissue cells increased between 7 and 180 days of age and was reduced in older animals. The 180-day-old animals presented a well-developed pericellular matrix rich in proteoglycans. The mean diameter of collagen fibrils increased from 7 to 180 days and was the same at 1,080 days. Glycosaminoglycan content was higher in 7-day-old animals. A higher amount of hydroxyproline was observed at 180 and 1,080 days. The swelling test showed a significant increase of wet weight in 7-day-old animals. In conclusion, the alterations that occur in the pressure-bearing of bullfrog PLT are the result of physiological alterations of the animal with the maturation and aging.

Distribution and innervation of putative arterial chemoreceptors in the bullfrog (Rana catesbeiana).

Peripheral arterial chemoreceptors have been located previously in the carotid labyrinth, the aortic arch, and the pulmocutaneous artery of frogs. In the present study we used cholera toxin B neuronal tract tracing and immunohistochemical markers for cholinergic cells (vesicular acetylcholine transporter [VAChT]), tyrosine hydroxylase (TH), and serotonin (5HT) to identify putative O2-sensing cells in Rana catesbeiana. We found potential O2-sensing cells in all three vascular areas innervated by branches of the vagus nerve, whereas only cells in the carotid labyrinth were innervated by the glossopharyngeal nerve. Cells containing either 5HT or TH were found in all three sites, whereas cells containing both neurotransmitters were found only in the carotid labyrinth. Cell bodies containing VAChT were not found at any site. The morphology and innervation of putative O2-sensing cells were similar to those of glomus cells found in other vertebrates. The presence of 5HT- and TH-immunoreactive cells in the aorta, pulmocutaneous artery, and carotid labyrinth appears to reflect a phylogenetic transition between the major neurotransmitter seen in the putative O2-sensing cells of fish (5HT) and those found in the glomus cells of mammals (acetylcholine, adenosine, and catecholamines).

Ultrastructure of the bullfrog (Lithobates catesbeianus-Shaw, 1802) oviduct in different seasons.

The bullfrog (Lithobates catesbeianus) is an exotic animal that adapted perfectly to the climatic conditions of Brazil after introduction in the country. These climatic conditions favor the reproduction and fattening of bullfrogs. However, the development of the bullfrog is significantly reduced in colder regions because low temperatures affect the secretion of gonadotropins and change the sensitivity of the germinal epithelium to gonadotropic hormones. Thus, the temperature and photoperiod are abiotic factors that influence the life cycle of this species, especially with regard to reproduction. Samples from different regions of the oviducts (the infundibulum, magnum, and isthmus) of 9 bullfrogs were collected in the different seasons of the year. The samples were fixed and processed for analysis with scanning and transmission electron microscopy. A comparison of the morphology of the oviducts collected during different seasonal periods suggests that the frogs have greater reproductive potential in the spring due to the development of the structures related to the oviduct, presence of developed cilia, and electron-dense granules. However, seasonal changes were noted in the animal throughout the year in preparation for reproduction during the spring and summer.

Distribution of somatostatin receptor 5 in mouse and bullfrog retinas.

Somatostatin (SRIF), as a neuroactive peptide in the CNS, may act as a neuromodulator through activation of five specific receptor subtypes (sst(1)-sst(5)). In this work we conducted a comparative study of the expression of sst(5) in mouse and bullfrog retinas by immunofluorescence double labeling. Basically, the expression profiles of sst(5) in the retinas of the two species were similar. That is, in the inner retina sst(5) was localized to dopaminergic and cholinergic amacrine cells, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, and cells in the ganglion cell layer, whereas in the outer retina immunostaining for sst(5) was observed in horizontal cells. However, a more widespread, abundant distribution of labeling for sst(5), as compared to mouse retina, was seen in bullfrog retina: strong labeling for sst(5) was diffusely distributed in both outer and inner plexiform layers (OPL and IPL) in the bullfrog retina, but the labeling was only observed in the IPL of the mouse retina. In addition, bullfrog photoreceptors, both rods and cones, but not mouse ones, were labeled by sst(5). In combination with the experiments showing that SRIF-immunoreactivity was mainly found in the inner retina, our results suggest that SRIF, released from SRIF-containing cells in the inner retina, may play a neuromodulatory role in both outer and inner retina mediated by volume transmission via sst(5) in bullfrog retina, while the SRIF action may be largely restricted to the mouse inner retina.

Interspecific comparisons of lymph volume and lymphatic fluxes: do lymph reserves and lymph mobilization capacities vary in anurans from different environments?

The femoral lymph sac volumes and lymph mobilization capacity were compared in three anuran species that span a range of environments, dehydration tolerance, ability to maintain blood volume with dehydration, and degrees of development of skeletal muscles putatively involved in moving lymph vertically to the posterior lymph hearts. The femoral lymph sac volume determined by Evans blue injection and dilution in the femoral lymph sac varied interspecifically. The semiaquatic species, Lithobates catesbeianus, had the greatest apparent lymph volume expressed either as 18.7 mL kg body mass⁻¹ or 94 mL kg thigh mass⁻¹, compared with both the terrestrial and aquatic species, Rhinella marina (7.3 mL kg body mass⁻¹ and 57 mL kg thigh mass⁻¹) and Xenopus laevis (6.5 mL kg body mass⁻¹ and 40 mL kg thigh mass⁻¹, respectively. Injections of Evans blue into the subvertebral lymph sac, which communicates with both pairs of lymph hearts, yielded the highest rates of lymph return to the circulation in all three species. The most terrestrial species had a greater rate of lymphatic return from the subvertebral lymph sac, compared with the other two species. The rate of lymph flux from the femoral sac varied interspecifically and was correlated with the number and development of skeletal muscles involved in lymph movement. The results indicated that the three species differ in both the volume of lymph present and the capacity to return lymph. Lymph flux was correlated with habitat and the ability to maintain blood volume when challenged by dehydration or hemorrhage, whereas femoral lymph volume was not correlated with these factors.

Allometry and sexually dimorphic traits in male anurans.

Allometry of secondary sexual traits has been the subject of recent debate, and the generality of positive allometry and its association with sexual selection have been recently questioned. Whereas some studies suggest an almost universal positive allometry for traits under sexual selection and isometry or a negative allometry for traits not under such pressure, other studies argue that this pattern results from the study of exaggerated (ornamental) traits. To answer the call for an examination of the allometry of less-exaggerated sexually selected traits, we have examined morphological data from 14 sexually dimorphic traits and six monomorphic traits from three anuran species. Although we found evidence of positive allometry in male secondary sexual traits of several species and populations, not all nonsexual traits were isometric or exhibited negative allometry. Furthermore, our results indicate that larger traits in the populations that we studied were not associated with greater allometric slopes. Therefore, our study is in line with the contention suggesting no specific kind of allometric pattern for sexual and nonsexual characters, and we can only advocate for further investigation of trait allometry and sexual selection to understand the complexity underlying the evolution of allometry in sexual traits.

Estudio morfológico, morfométrico e histoquímico de los epitelios de revestimiento y glandular de la lengua de la rana toro, rana catesbeiana / Morphological, morphometrical and histochemical study of the lining and glandular epithelia of the tongue of the tullfrog rana catesbeiana

La superficie dorsal de la lengua de la rana toro, Rana catesbeiana, presenta un epitelio simple cilíndrico, constituido por células caliciformes y raras células ciliadas. El dorso de la lengua posee numerosas papilas filiformes y algunas fungiformes. Las primeras poseen un epitelio simple cilíndrico, con células secretoras, mientras que las segundas poseen en la región apical, un disco sensorial con epitelio estratificado cilíndrico, con células basales, periféricas, glandulares y receptoras. A lo largo del dorso de la lengua existen numerosas glándulas tubulares, que penetran en profundidad, entremezclándose con las fibras musculares. El epitelio glandular es simple cilíndrico, con células secretoras y de sostén. Las primeras son las únicas en la base de la glándula y las segundas solo se encuentran en número escaso en el tercio superior. La superficie ventral de la lengua posee un epitelio estratificado, con células caliciformes y, entre éstas, células ciliadas. La morfometría de las glándulas mostró que son más cortas en la región anterior de la lengua (330 um) que en la región posterior (450 um). Las células secretoras de las glándulas linguales anteriores son menores (1457,7 um3) que en las posteriores (2645,9 um3). Lo mismo ocurre con los núcleos celulares: 130,0 um3 en las glándulas anteriores y 202,3 um3 en las posteriores. Las células secretoras de las glándulas linguales sintetizan producto rico en proteínas y mucopolisacáridos neutros, pudiendo caracterizarse como seromucoso. Las células caliciformes de las superficies dorsal y ventral secretan proteínas y mucopolisacáridos neutros, clasificándose como del tipo G1, mientras que las células de sostén de las glándulas superficiales de las papilas fungiformes secretan moco rico en mucopolisacáridos neutros, sulfomucinas y sialomucinas.

The dorsal surface of the tongue of the bullfrog, Rana catesbeiana, has simple columnar epithelium with a few ciliated cells and goblet cells. The entire surface is covered with numerous filiform papillae and few fungiform. Filiform papillae have a simple columnar epithelium with secretory cells, while the fungiform have a sensory disc on their upper surface the lined by a stratified columnar epithelium with basal, peripheral, glandular and receptor cells. Over the dorsal lingual surface there are numerous winding tubular glands, which penetrate deeply into the muscle of the tongue, mingling with the fibers. The gland epithelium is cylindrical with secretory and supporting cells. The first are absolute on the basis of the gland and the latter are rare in the upper third. The ventral surface of the tongue is lined by a stratified epithelium, with the presence of goblet cells, with ciliated cells among them. Morphometrically, lingual glands varies in length, according to their location: shorter in the anterior region of the tongue (330 um) than in the posterior region (450 um). Secretory cells of the anterior lingual glands are smaller (1457.7 mm3) than the posterior ones (2645.9 um3). The same can be said of the cell nuclei, 130.0 um3 for the anterior glands and 202.3 um3 for the posterior ones. Secretory cells of the lingual glands contain substances rich in protein and neutral mucopolysaccharides, which characterize the seromucous type. Goblet cells of the dorsal and ventral surface epithelia secrete neutral mucopolysaccharides and proteins, and can be characterized as type G1 cells, and the supporting cells of the superficial glands of the fungiform papillae secrete a mucus rich in neutral mucopolysaccharides, sulfomucins and sialomucins.

Response properties and receptive field organization of collision-sensitive neurons in the optic tectum of bullfrog, Rana catesbeiana.

OBJECTIVE: Many studies have reported that animals will display collision avoidance behavior when the size of retinal image of an object reaches a threshold. The present study aimed to investigate the neural correlates underlying the frog collision avoidance behavior. METHODS: Different types of visual stimuli simulating the retinal image of an approaching or a recessing object were generated by a computer and presented to the right eye of frog. A multielectrode array was used to examine the activity of collision-sensitive neurons, and single electrode recordings were employed to quantify visual parameter(s) of the frog collision-sensitive neurons. RESULTS: The multielectrode array revealed that 40 neurons in the optic tectum showed selective responsiveness to objects approaching on a direct collision course. The response profiles of these collision-sensitive neurons were similar to those of lobula giant movement detector (LGMD) in the locust or to those of neurons in the pigeon. However, the receptive field (RF) size of the frog neurons [(18.5+/-3.8) degrees, n=33)] was smaller than those of collision-sensitive neurons of the locust and the pigeon. Multielectrode recordings also showed that the collision-sensitive neurons were activated only when the focus of expansion of a looming retinal image was located within the center of its RF. There was a linear relationship between the parameter l/v (l denotes half-size of the object, v denotes approaching velocity) and time-to-collision (time difference between the peak of the neuronal activity and the predictive collision) in 16 collision-sensitive neurons. Theoretical consideration showed that the peak firing rate always occurred at a fixed delay of (60.1 +/- 39.5) ms (n=16) after the object had reached a constant angular size of (14.8 +/- 3.4) degrees (n=16) on the retina. CONCLUSION: The results may help clarify the mechanisms underlying the collision avoidance behavior in bullfrog.

A neural basis for motor primitives in the spinal cord.

Motor primitives and modularity may be important in biological movement control. However, their neural basis is not understood. To investigate this, we recorded 302 neurons, making multielectrode recordings in the spinal cord gray of spinalized frogs, at 400, 800, and 1200 mum depth, at the L2/L3 segment border. Simultaneous muscle activity recordings were used with independent components analysis to infer premotor drive patterns. Neurons were divided into groups based on motor pattern modulation and sensory responses, depth recorded, and behavior. The 187 motor pattern modulated neurons recorded comprised 14 cutaneous neurons and 28 proprioceptive neurons at 400 mum in the dorsal horn, 131 intermediate zone interneurons from approximately 800 microm depth without sensory responses, and 14 motoneuron-like neurons at approximately 1200 microm. We examined all such neurons during spinal behaviors. Mutual information measures showed that cutaneous neurons and intermediate zone neurons were related better to premotor drives than to individual muscle activity. In contrast, proprioceptive-related neurons and ventral horn neurons divided evenly. For 46 of the intermediate zone interneurons, we found significant postspike facilitation effects on muscle responses using spike-triggered averages representing short-latency postspike facilitations to multiple motor pools. Furthermore, these postspike facilitations matched significantly in both their patterns and strengths with the weighting parameters of individual primitives extracted statistically, although both were initially obtained without reference to one another. Our data show that sets of dedicated interneurons may organize individual spinal primitives. These may be a key to understanding motor development, motor learning, recovery after CNS injury, and evolution of motor behaviors.

Development of tectal connectivity across metamorphosis in the bullfrog (Rana catesbeiana).

In the bullfrog (Rana catesbeiana), the process of metamorphosis culminates in the appearance of new visual and visuomotor behaviors reflective of the emergence of binocular vision and visually-guided prey capture behaviors as the animal transitions to life on land. Using several different neuroanatomical tracers, we examined the substrates that may underlie these behavioral changes by tracing the afferent and efferent connectivity of the midbrain optic tectum across metamorphic development. Intratectal, tectotoral, tectotegmental, tectobulbar, and tecto-thalamic tracts exhibit similar trajectories of neurobiotin fiber label across the developmental span from early larval tadpoles to adults. Developmental variability was apparent primarily in intensity and distribution of cell and puncta label in target nuclei. Combined injections of cholera toxin subunit ß and Phaseolus vulgaris leucoagglutinin consistently label cell bodies, puncta, or fiber segments bilaterally in midbrain targets including the pretectal gray, laminar nucleus of the torus semicircularis, and the nucleus of the medial longitudinal fasciculus. Developmentally stable label was observed bilaterally in medullary targets including the medial vestibular nucleus, lateral vestibular nucleus, and reticular gray, and in forebrain targets including the posterior and ventromedial nuclei of the thalamus. The nucleus isthmi, cerebellum, lateral line nuclei, medial septum, ventral striatum, and medial pallium show more developmentally variable patterns of connectivity. Our results suggest that even during larval development, the optic tectum contains substrates for integration of visual with auditory, vestibular, and somatosensory cues, as well as for guidance of motivated behaviors.

Mechanical properties of the hindlimb bones of bullfrogs and cane toads in bending and torsion.

When compared with most vertebrates, frogs use a novel style of jumping locomotion powered by the hindlimbs. Hindlimb bones of frogs must withstand the potentially erratic loads associated with such saltatory locomotion. To evaluate the load bearing capacity of anuran limb bones, we used three-point bending, torsion, and hardness tests to measure the mechanical properties of the femur and tibiofibula from adults of two species that use different jumping styles: explosively jumping bullfrogs (Rana (Lithobates) catesbeiana) and cyclically hopping cane toads (Bufo (Chaunus) marinus). Yield stress and strain values for R. catesbeiana and B. marinus hindlimb bones are within the range of values previously reported for other vertebrates. However, anuran hindlimb bones generally stand out as having higher yield stresses in bending than those of closely related, nonsaltatory salamanders, highlighting the importance of considering phylogenetic context in comparisons of bone functional capacity and adaptation. Stiffness values for both frog species tested were also high, which may facilitate efficient transmission of muscular forces while jumping. Elevated stiffness may also contribute to some discrepancies between determinations of bone properties via hardness versus bending tests. In comparisons between species, B. marinus bones showed significantly higher bending yield stresses than R. catesbeiana, whereas R. catesbeiana bones showed significantly higher torsional yield stresses than B. marinus. These differences may correlate with differences in jumping style and limb anatomy between ranid and bufonid frogs, suggesting that evolutionary changes in bone mechanical properties may help to accommodate new functional demands that emerge in lineages.

Localization of hematopoietic cells in the bullfrog (Lithobates catesbeianus).

Amphibians represent the first phylogenetic group to possess hematopoietic bone marrow. However, adult amphibian hematopoiesis has only been described in a few species and with conflicting data. Bone marrow, kidney, spleen, liver, gut, stomach, lung, tegument, and heart were therefore collected from adult Lithobates catesbeianus and investigated by light microscopy and immunohistochemical methods under confocal laser microscopy. Our study demonstrated active hematopoiesis in the bone marrow of vertebrae, femur, and fingers and in the kidney, but no hematopoietic activity inside other organs including the spleen and liver. Blood cells were identified as a heterogeneous cell population constituted by heterophils, basophils, eosinophils, monocytes, erythrocytic cells, lymphocytes, and their precursors. Cellular islets of the thrombocytic lineage occurred near sinusoids of the bone marrow. Antibodies against CD34, CD117, stem cell antigen, erythropoietin receptor, and the receptor for granulocyte colony-stimulating factor identified some cell populations, and some circulating immature cells were seen in the bloodstream. Thus, on the basis of these phylogenetic features, we propose that L. catesbeianus can be used as an important model for hematopoietic studies, since this anuran exhibits hematopoiesis characteristics both of lower vertebrates (renal hematopoiesis) and of higher vertebrates (bone marrow hematopoiesis).

Distribution of frequencies of spontaneous oscillations in hair cells of the bullfrog sacculus.

Under in vitro conditions, free-standing hair bundles of the bullfrog (Rana catesbeiana) sacculus have exhibited spontaneous oscillations. We used a high-speed complementary metal oxide semiconductor camera to track the active movements of multiple hair cells in a single field of view. Our techniques enabled us to probe for correlations between pairs of cells, and to acquire records on over 100 actively oscillating bundles per epithelium. We measured the statistical distribution of oscillation periods of cells from different areas within the sacculus, and on different epithelia. Spontaneous oscillations exhibited a peak period of 33 ms (+29 ms, -14 ms) and uniform spatial distribution across the sacculus.

Effect of gap junction blocker beta-glycyrrhetinic acid on taste disk cells in frog.

A gap junction blocker, 18beta-glycyrrhetinic acid (beta-GA), increased the membrane resistance of Ia, Ib and II/III cells of frog taste disk by 50, 160, and 300 M Omega, respectively, by blocking the gap junction channels and hemichannels. The amplitudes of gustatory depolarizing potentials in the disk cells for 4 basic taste stimuli were reduced to 40-60% after intravenous injection of beta-GA at 1.0 mg/kg. beta-GA of 1.0 mg/kg did not affect the resting potentials and the reversal potentials for tastant-induced depolarizing potentials in any taste disk cells. The percentage of cells responding to each of 4 basic taste stimuli and varying numbers of 4 taste qualities did not differ between control and beta-GA-treated taste disk cells. This implies that gustatory depolarizing response profiles for 4 basic taste stimuli were very similar in control and beta-GA-treated taste disk cells. It is concluded that beta-GA at 1.0 mg/kg reduced the amplitude of gustatory depolarizing potentials in taste disk cells by strongly blocking depolarizing currents flowing through the gap junction channels and hemichannels, but probably weakly affected the gustatory transduction mechanisms for 4 taste stimuli.

Study design, water quality, morphometrics and age of the bullfrog, Rana catesbeiana, in sub-watersheds of the Yamaska River drainage basin, Québec, Canada.

The Yamaska River basin is renowned for its poor water quality, which has been attributed to intensive agriculture (corn, soya, high-density pork and poultry production). Six locations within the Yamaska watershed were selected to evaluate the impact of agriculture on water habitats and study the bullfrog as a sentinel species of potential exposure and effects. The selected sub-watersheds were chosen according to the percentage of surface area under cultivation and classified as low (0-19%), moderate (20-59%) or high (>60%). In 2004 and 2005, analysis of surface water samples demonstrated that pesticide concentrations and most water quality parameters increased with increasing agricultural activity. Sixteen adult bullfrogs were sampled from each site. Animals were weighed, measured for length (total, snout-vent, tibia, tympanum), sexed, and evaluated for the colour of the throat. Skeletochronology was used to estimate the growth and age of the frogs. Cross-sections of decalcified phalanges and femurs were treated in order to count LAGs (lines of arrest growth) and for the observations of other parameters related to bone growth. The bullfrogs from highly contaminated sites had the lowest mean age and the smallest snout-vent length compared to sites of low contamination.