Symbiotic fermentation, digesta passage, and gastrointestinal morphology in bullfrog tadpoles (Rana catesbeiana)

Relative to other herbivorous vertebrates, the nutritional ecology and digestive physiology of anuran larvae remain poorly understood. Our objective was to compare gut structure and inhabitants, digesta passage, and microbial fermentation in bullfrog tadpoles (Rana catesbeiana) to those in other herbivores. Bullfrog tadpole gastrointestinal tracts were long and voluminous, with an enlarged colon that harbored a diverse symbiotic community. The transit time for particulate markers passing through bullfrog tadpoles was 6 h, the median retention time was 8-10 h, and gut clearance was 10-14 h postingestion. Relatively high levels of short-chain fatty acids in the hindgut of tadpoles indicated active microbial fermentation in this gut region. This report represents the first account of gastrointestinal fermentation in the class Amphibia. On the basis of in vitro fermentation assays, we estimated that microbial fermentation in the hindgut provides 20% of the total daily energy requirement of bullfrog tadpoles. These tadpoles also exhibited coprophagy, a practice that provides important nutritive gains in other herbivores. The physiological and behavioral characteristics of these tadpoles are remarkably similar to those of other small-bodied, hindgut-fermenting vertebrates, suggesting convergent digestive strategies among a broad range of herbivorous taxa.     

Expanded geographical range, new host accounts, and observations of the nematode Gyrinicola batrachiensis (Oxyuroidea: Pharyngodonidae) in tadpoles

We describe the first account of the nematode Gyrinicola batrachiensis in the gastrointestinal tracts of tadpoles of Bufo terrestris, Gastrophryne carolinensis, Hyla femoralis, H. squirella, Rana heckscheri, and R. utricularia. Infection by G. batrachiensis was also noted in tadpoles of R. catesbeiana and R. clamitans, species previously described as harboring these nematodes. These observations represent a major expansion of the known geographical range of G. batrachiensis to include the southeastern United States. In our study, female G. batrachiensis nematodes in ranid host species were didelphic, but females in B. terrestris tadpoles were monodelphic. Furthermore, only female nematodes were found in the latter host species. Such variations in nematode uterine morphology and sex ratios among these tadpole host species are in accordance with the apomictic and haplodiploid reproductive strategies previously reported for G. batrachiensis. These observations also support a generalized pattern relating G. batrachiensis reproductive modes and the life cycles of their tadpole hosts.     

Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana)

Summary Vascular adrenergic sensitivity to exogenous catecholamines was examined in tadpoles of the American bullfrog (Rana catesbeiana), ranging from stage III to XIV. Central arterial blood pressure was measured in decerebrate bullfrog tadpoles to determine a reasonable initial infusion pressure. Solutions of epinephrine and phenylephrine were infused into the vasculature of pithed tadpoles, and the resulting changes in vascular resistance (R _v) were used to construct log dose-response relationships. Epinephrine infusion produced a dose-dependent increase in R _v (EC₅₀=5.3·10^-7 M), which could be reversed by sodium nitroprusside (a smooth muscle relaxant) and blocked by phenoxybenzamine (an -adrenergic antagonist). Larval R _v also increased with infusion of the -agonist phenylephrine (EC₅₀=7.4·10⁸ M). Infusion of 10^-6 M isoproterenol (a -agonist) largely reversed the phenylephrine-induced increase in R _v. These results indicate that the capacity exists for both -mediated vasoconstriction and -mediated vasodilation early in bullfrog ontogeny. Neither initial R _v nor the responses to infused epinephrine or phenylephrine were significantly correlated to development over the range of larval stages used in this study.Abbreviations ECG electrocardiogram - EPI epinephrine - ISO isoproterenol - PHE phenylephrine - POB phenoxybenzamine - R _v vascular resistance - SNP sodium nitroprusside     

Pharmacokinetics of kanamycin in the bullfrog, Rana catesbeiana

1. Kanamycin disposition was studied in bullfrogs (Rana catesbeiana) following single doses IP. Both plasma t1/2 and Vd of the drug increased with increasing time after drug indicating redistribution and tight binding of kanamycin to deep tissue compartments. 2. Kanamycin was eliminated unchanged with a t1/2 plasma = 27 hr; perilymph = 89 hr; endolymph = 183 hr; aqueous humor = 54 hr; and CSF = 58 hr. 3. Kanamycin was absorbed by frogs from environmental water. 4. Environmental conditions must be carefully specified and monitored, as well as the physiological state of the animals when studying the effects of drugs on Amphibia.     

Primary structure of a ribonuclease from bullfrog (Rana catesbeiana) liver

A pyrimidine base-specific ribonuclease was purified from bullfrog (Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys65-Cys126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani et al. [Biochemistry (1988) 26, 2189-2194] and R. japonica egg by Kamiya et al. [Seikagaku (in Japanese) (1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.     

Genetic evidence for female-biased dispersal in the bullfrog, Rana catesbeiana (Ranidae)

Sex-biased dispersal is an important but unexplored area of amphibian ecology. We predicted female-biased dispersal in the bullfrog (Rana catesbeiana) based on aspects of their mating system and tested this prediction using data from seven polymorphic DNA microsatellite loci. Allelic (F-statistics) and genotypic [assignment index, (AIc)] frequencies from nine Ontario populations support our prediction, although significant sex differences in inbreeding and variance of AIc were not detected. The diversity of mating systems found in amphibians represents an important avenue for investigating the relationship between reproductive systems, dispersal and phylogeny.     

Embryonic and larval hemoglobins during the early development of the bullfrog, Rana catesbeiana

The main hemoglobin (Hb) found in Shumway (embryonic) stage 25 bullfrogs is that which we have designated Td-4. The other major tadpole Hbs (Td-1, 2, and 3) predominate during Taylor and Kollros (larval) stages I-XVIII. We propose that Td-4 is an embryonic Hb, whereas Td-1, 2, and 3 are larval (fetal-like) Hbs. Embryonic Hb Td-4 continues to be synthesized during the larval stages. During the larval period, the average peripheral blood Hb profile changes very little with morphological stage or general growth. However, there is great heterogeneity in the embryonic:larval Hb ratio among individual tadpoles of a given stage or weight, apparently due to differential Hb and red cell production by the two active erythropoietic sites, mesonephric kidneys (Td-4), and liver (Td-1, 2, 3).     

Biomechanics of vibration reception in the bullfrog,Rana catesbeiana

The opercularis system (OPS) of amphibians consists of an opercularis muscle that connects the shoulder girdle skeleton to the operculum, a movable element in the oval window of the otic capsule. The role of the OPS in reception of vibrations was examined in bullfrogs (Rana catesbeiana) tested in various postures that manipulated differential motion between the shoulder girdle (the origin of the opercularis muscle) and skull (including the inner ear). Amplitude and phase relationship of motions of the suprascapular cartilage of the shoulder girdle and the posterior skull were also measured during these tests. 1. Microphonic responses to vertical vibrations from 25-200 Hz were typically highest when frogs were in a normal, sitting posture with the head held off the vibrating platform. Responses from animals in which the head directly contacted the platform were often less (by up to 10 dB at certain frequencies). Responses from all test positions were highest at lower frequencies, especially between 50-100 Hz. 2. Suprascapular accelerations were typically highest in the normal, sitting posture, and at lower frequencies (50-75 Hz) were often greater than that of the vibrating platform by up to 8 dB. The shoulder girdle skeleton of the bullfrog is therefore readily affected by vertical substrate motion. 3. The amplitude of microphonic responses in the different test postures did not correspond well with head acceleration. Rather, response amplitude corresponded best with the absolute difference between shoulder and head motion. For example, in the normal posture, suprascapular motion was much greater than head motion, and responses were relatively high. If only the head was vibrated, head motion was high and shoulder motion low, and responses also were relatively high. If the head and body were vibrated together, their motions were similar, and responses to the same platform accelerations were often reduced. Phase differences between shoulder and head motions were small at the frequencies examined and may be of little functional significance. The importance of differences in shoulder and head motion suggests that the resulting differential motion of the operculum and inner ear fluids can produce waves that stimulate appropriate end organs (such as the saccule). 4. Removal of the opercularis muscle reduced responses up to 18 dB at certain frequencies in some of the test postures. The most significant reductions were observed in those postures with a significant difference between shoulder and head motion (such as the normal posture).(ABSTRACT TRUNCATED AT 400 WORDS)     

The decrease in plasma melatonin at metamorphic climax in Rana catesbeiana (bullfrog) tadpoles is induced by thyroxine.

Melatonin decreases in the plasma of Rana catesbeiana (bullfrog) tadpoles at the climax of metamorphosis when the thyroxine (T(4)) level peaks. Since melatonin inhibited thyroid function in vitro, it would be of interest to determine if the decline in plasma melatonin permits greater thyroid hormone secretion, or if the increasing levels of T(4) cause the climactic decrease in plasma melatonin. The reciprocal effects of administering T(4) or melatonin just prior to metamorphic climax were examined in tadpoles kept at 22 degrees C on an 18L:6D cycle. If melatonin functions as a thyroid antagonist at later metamorphic stages, administration of melatonin should decrease plasma T(4), whereas if T(4) causes the decline in plasma melatonin, T(4) treatment of tadpoles prior to climax should induce the climactic melatonin decrease prematurely. Once daily injection of 40 microg melatonin for 5 days at 19.30 h had no effect on metamorphic progress, or on plasma T(4) or melatonin levels, except for a transient rise in melatonin just after the injection. Immersion in 2.2x10(-4) M melatonin for 6 days accelerated metamorphosis and decreased plasma melatonin, but had no effect on plasma T(4). Administration of T(4) by injection of 0.2 microg, or immersion in a 6.3x10(-8) M solution accelerated metamorphosis more than melatonin immersion, raised plasma T(4) to climax levels, and induced a decrease in plasma melatonin. We conclude that rapid clearance of exogenous melatonin from the circulation in these experiments did not allow it to affect plasma T(4), and that there is clear evidence that the rise in T(4) induces the climax decrease in plasma melatonin. The finding that immersion in a high level of melatonin can lower plasma melatonin and accelerate metamorphosis, whereas a single daily injection does not, provides an explanation for some of the contradictory reports in the literature concerning melatonin's effect on tadpole metamorphic progress.     

The tactile-stimulated startle response of tadpoles: acceleration performance and its relationship to the anatomy of wood frog (Rana sylvatica), bullfrog (Rana catesbeiana), and American toad (Bufo americanus) tadpoles

I described the tactile-stimulated startle response (TSR) of wood frog (Rana sylvatica), bullfrog (Rana catesbeiana), and American toad (Bufo americanus) tadpoles. One purpose was to rank species in terms of maximum acceleration performance. Also, I tested whether anatomical indicators of performance potential were predictive of realized performance. TSRs were elicited in a laboratory setting, filmed at 250 Hz, and digitally analyzed. TSRs began with two, initial body curls during which tadpoles showed a broad spectrum of movement patterns. TSR performance was quantified by maximum linear acceleration and maximum rotational acceleration of the head/body, both of which tended to occur immediately upon initiation of motion (< 0.012 sec into the response). Bullfrog tadpoles had higher maximum acceleration than the other species, but other interspecific differences were not significant. The species' rank order for the anatomical indicator of linear acceleration potential was bullfrog > wood frog > American toad. The species' rank order for the anatomical indicator of rotational acceleration potential was bullfrog > wood frog = American toad. Thus, the anatomical indicators roughly predicted the rank order of interspecific average performance. However, the anatomical indicators did not correlate with individual tadpole performance. Variability in behavioral patterns may obscure the connection between anatomy and performance. This is seen in the current lack of intraspecific correlation between a morphological indicator of acceleration capacity and acceleration performance.     

Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog)

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, ~50 and ~25%, respectively.     

Effects of feeding on metabolism, gas transport, and acid-base balance in the bullfrog Rana catesbeiana

Massive feeding in ectothermic vertebrates causes changes in metabolism and acid-base and respiratory parameters. Most investigations have focused on only one aspect of these complex changes, and different species have been used, making comparison among studies difficult. The purpose of the present study was, therefore, to provide an integrative study of the multiple physiological changes taking place after feeding. Bullfrogs (Rana catesbeiana) partly submerged in water were fed meals (mice or rats) amounting to approximately (1)/(10) of their body weight. Oxygen consumption increased and peaked at a value three times the predigestive level 72-96 h after feeding. Arterial PO(2) decreased slightly during digestion, whereas hemoglobin-bound oxygen saturation was unaffected. Yet, arterial blood oxygen content was pronouncedly elevated because of a 60% increase in hematocrit, which appeared mediated via release of red blood cells from the spleen. Gastric acid secretion was associated with a 60% increase in plasma HCO3(-) concentration ([HCO3(-)]) 48 h after feeding. Arterial pH only increased from 7.86 to 7.94, because the metabolic alkalosis was countered by an increase in PCO(2) from 10.8 to 13.7 mm Hg. Feeding also induced a small intracellular alkalosis in the sartorius muscle. Arterial pH and HCO3(-) returned to control values 96-120 h after feeding. There was no sign of anaerobic energy production during digestion as plasma and tissue lactate levels remained low and intracellular ATP concentration stayed high. However, phosphocreatine was reduced in the sartorius muscle and ventricle 48 h after feeding.     

Evidence concerning the morphogenesis of saccular receptors in the bullfrog (Rana catesbeiana)

A dichotomy of hair-cell types has been found in the bullfrog sacculus, and considerable evidence supports the view that one type (“peripheral”) is transformed during macular growth to the other type (“central”). Between the periphery and the center of the macula, one finds a gradation of form from “peripheral” to “central” type. Occasionally in adults and more often in stage-26 tadpoles one finds the presumably younger peripheral type of hair cell occurring well beyond the limits of the macula proper. The apparent morphogenic sequence for saccular hair cells is (1) development of a kinocillum on an endolymphatic epithelial cell, (2) gradual transformation of microvilli into stereocilia, (3) growth of the stereocilia and development of kinociliary bulb, (4) achievement of final size and form.     

Characterization of cholecystokinin receptors in bullfrog (Rana catesbeiana) brain and pancreas

The binding of biologically active 125I-Bolton-Hunter-CCK-33 to bullfrog brain and pancreatic membrane particles was characterized. Both tissues exhibited time-dependent, saturable, reversible, and high affinity binding without evidence for cooperative interaction. Both bullfrog CCK receptors resembled their mammalian counterparts in having acidic pH optima for tracer binding and a Kd of about 0.5 nM. However, the receptors differed from their mammalian counterparts in that (1) the bullfrog brain membranes bound more tracer per mg protein than did the pancreatic membranes, (2) both bullfrog CCK receptors were relatively insensitive to dibutyryl cGMP, and (3) both bullfrog brain and pancreatic CCK receptors exhibited the same general specificity toward a variety of CCK and gastrin peptides. For both tissues, the relative order of receptor binding potency was CCK-8 greater than caerulein = CCK-33 greater than gastrin-17-II greater than CCK-8-ns = gastrin-17-I greater than caerulein-ns greater than gastrin-4 with the sulfated CCK peptides being 1000-fold more potent than their nonsulfated analogs. Sulfated gastrin was also relatively potent, being only 10-fold weaker than CCK-8. Gastrin-4 was 20 000-fold weaker than CCK-8 in interacting with the brain CCK receptor. The latter finding is in sharp contrast to the mammalian brain CCK receptor. We conclude that the bullfrog brain and pancreas contain similar CCK receptors of probable physiological significance and may represent an ancestral condition from which the two distinct CCK receptors present in mammalian brain and pancreas have evolved.     

Sauvagine-like and corticotropin-releasing factor-like immunoreactivity in the brain of the bullfrog (Rana catesbeiana)

Summary Immunocytochemical methods were used to investigate the occurrence and distribution of sauvagine, corticotropin-releasing factor-, or urotensin I-like immunoreactivities (SVG-ir, CRF-ir, UI-ir, respectively) in the bullfrog (Rana catesbeiana) brain, using specific antisera raised against non-conjugated SVG, ovine CRF, rat/human CRF, and UI. In the hypothalamus, SVG-ir was found in the magnocellular perikarya, in the dorsal and ventral regions of the preoptic nucleus, and in the hypothalamo-hypophyseal projections to the external zone as well as the internal zone of the median eminence, to pars nervosa, and in fibres running from the pars nervosa to the pars intermedia of the pituitary. In contrast, CRF-ir was found only in parvocellular perikarya, mainly localized in the rostro-ventral part of the preoptic nucleus, with fine processes protruding through the ependyma of the third ventricle, fibre projections terminating in the anterior preoptic area and in the neuropil of the periventricular gray, and a caudal projection to the external zone of the median eminence. No CRF-ir staining was seen in the pars nervosa and pars intermedia. The use of UI-specific antisera failed to give a positive response in the frog brain. It is concluded that, in the frog brain, two anatomically different CRF-like (or SVG-like) systems co-exist, comparable to the reported co-existence of UI-ir and CRF-ir neuronal systems in fish brain.