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The husbandry and care of dendrobatid frogs.

Dendrobatid frogs are studied primarily for the bioactive alkaloids found in their skin. Also known as poison-dart frogs, these animals accumulate toxic alkaloids from dietary sources. The function and uses of the many alkaloids, the alkaloid accumulation system, and the basic biology and physiology of the frogs themselves are of research interest. Here we overview the taxonomy of these frogs and some of the unique aspects of their natural biology and reproduction. We also describe the components of a successful laboratory housing system, including temperature, lighting, humidity, ventilation, nutrition, health considerations, and handling. A brief summary of dendrobatid research highlights is provided.

Animal Husbandry↗

[Evaluation of the state of the gene pool of natural populations of vertebrate animals inhabiting the fragmented landscape of Moscow and Moscow district (with special reference to brown frogs)].

A system of urban ecological genetic monitoring has been developed for the first time in Russia and in the world. As a model, two species of brown frogs, Rana arvalis Nilss. and R. temporaria L. (16 populations, seven isozyme loci) were used. An evaluation of the gene pool state in urban frog populations has shown that the diversity in the Moscow frog populations was lower than that in natural populations (up to 80 and 50% in respectively R. anvalis and R. temporaria). Mean heterozygosities per locus were higher in large natural populations than in small urban isolates: in R. arvalis, these values were 0.16 and 0.06; in R. temporaria, 0.34 and 0.18, respectively. The number of polymorphic loci was also higher in natural populations than in the urban ones: 4 versus 2 in R. arvalis and 5 versus 4 in R. temporaria. Using superoxiddismutase as an example, fixation of different alleles of the same locus in different small isolates was shown. The gene pool condition of all but one urban populations of brown frog was evaluated as unsatisfactory, and that of the R. arvalis populations, as critical. These changes of the gene pool are explained mainly by gene drift accompanied by inbreeding, which was caused by human-induced fragmentation of the range and a decrease in population size of the species. The results of this study was employed in the development of the Moscow governmental program on restoration of the gene pools of vanishing animal species on specially protected natural urban territories. The series of works on long-term monitoring and assessment of the state of natural populations of model species in anthropogenic landscapes of Moscow and Moscow district has laid a foundation for a new branch of science, gene urbanology.

Alleles↗

Distribution of a putative kainic acid receptor in the frog central nervous system determined with monoclonal and polyclonal antibodies: evidence for synaptic and extrasynaptic localization.

A frog brain kainic acid receptor (KAR) was studied using monoclonal and polyclonal antibodies against the affinity-purified receptor. Immunocytochemistry was done on sections of the frog CNS, and the distribution of immunostaining was compared with the distribution of high- and low-affinity 3H-kainic acid (3H-KA) binding sites determined with in vitro receptor autoradiography. These studies showed (1) similar distributions of high- and low-affinity 3H-KA binding sites, (2) identical patterns of immunostaining with the polyclonal antibodies and 2 monoclonal antibodies, and (3) an antibody binding distribution which closely matched that of 3H-KA binding, suggesting that the antibodies recognize the primary KAR in frog brain. In the frog brain, an anteroposterior gradient of immunostaining was observed, with the telencephalon intensely and uniformly immunoreactive. Other areas intensely immunoreactive included the cerebellum, the infundibulum, the tectal and posterior commissures, and the laminar nucleus of the torus semicircularis. The optic tectum showed selective staining of the plexiform layers 3 and 5-7. The pattern of staining was punctate and appeared to be associated with nerve fibers, among them dendritic arborizations. Electron microscopic observations showed staining at the cytoplasmic side of postsynaptic membranes. Extra-synaptic staining was observed as patches on the surface of unmyelinated nerve processes.

Animals↗

Geographic variation of genetic and behavioral traits in northern and southern tüngara frogs.

We use a combination of microsatellite marker analysis and mate-choice behavior experiments to assess patterns of reproductive isolation of the túngara frog Physalaemus pustulosus along a 550-km transect of 25 populations in Costa Rica and Panama. Earlier studies using allozymes and mitochondrial DNA defined two genetic groups of túngara frogs, one ranging from Mexico to northern Costa Rica (northern group), the second ranging from Panama to northern South America (southern group). Our more fine-scale survey also shows that the northern and southern túngara frogs are genetically different and geographically separated by a gap in the distribution in central Pacific Costa Rica. Genetic differences among populations are highly correlated with geographic distances. Temporal call parameters differed among populations as well as between genetic groups. Differences in calls were explained better by geographic distance than by genetic distance. Phonotaxis experiments showed that females preferred calls of males from their own populations over calls of males from other populations in about two-thirds to three-fourths of the contrasts tested. In mating experiments, females and males from the same group and females from the north with males from the south produced nests and tadpoles. In contrast, females from the south did not produce nests or tadpoles with males from the north. Thus, northern and southern túngara frogs have diverged both genetically and bioacoustically. There is evidence for some prezygotic isolation due to differences in mate recognition and fertilization success, but such isolation is hardly complete. Our results support the general observation that significant differences in sexual signals are often not correlated with strong genetic differentiation.

Animals↗

Calling is an honest indicator of paternal genetic quality in poison frogs.

Several competing hypotheses have been put forward to explain why females of many species mate preferentially with males possessing the most conspicuous signals (e.g., ornaments, displays, or songs). We performed a laboratory experiment using two species of poison frogs, Dendrobates leucomelas and Epipedobates tricolor, to test the hypothesis that male calling performance is an honest indicator of parental quality. Our analyses are based on data from behavioral observations of mating activities of captive-reared individuals (and their offspring) that were housed in terraria for four consecutive breeding seasons. Male mating success increased with male calling rate and chirp duration in both species, suggesting that females preferred males with more elaborate calls. Because calling performance improved with age in D. leucomelas, female poison frogs that prefer males with more elaborate calls in the wild may end up mating with older males that have already proven their ability to survive. Females that mated with good callers obtained higher quality offspring. Eggs fertilized by males with high calling rates and long chirp durations had higher hatching success and produced tadpoles that were more likely to metamorphose into surviving frogs. As a consequence, females that mated with males with high calling performance obtained more surviving offspring per egg, compared to females that mated with poor callers. Collectively, our findings comply with the notion that female poison frogs prefer to mate with good callers because calling performance is a reliable predictor of offspring quality. The possible influence of maternal allocation and reasons for the strong effect size compared to previous studies are discussed.

Aging↗

[Effects of anti-cholinoceptor antibodies on the frog heart].

Research on isolated hearts of Rana temporaria has shown that upon treating them with anti-cholinoreceptor antibodies, there occurs a considerable reduction of inhibition effect on cardiac activity of acetylcholine or carbacholine. A reduction of inhibition effect was noticed after incubation of frog's heart with antibodies against cholinoreceptors obtained from motor-denervated muscles of frogs as well as from muscles of mice Balb/c. Cholinoreceptor protein was obtained and purified by A. Sobel's method. Control tests were made with serum of non-immunized rabbits and rabbits immunized with material obtained from non-denervated muscles of frogs. It was concluded that acetylcholinoreceptor antibodies are capable of provoking atropine-like effect on frog's heart. According to our data, anti-cholinoreceptor antibodies as well as cholinoreceptors are relatively non-specific to species.

Acetylcholine↗

Number of mast cells in the harderian gland of the green frog, Rana esculenta: the annual cycle and its relation to environmental and hormonal factors.

The Harderian gland of the green frog contains mast cells. Their number shows annual variations, being more numerous in the winter months. The increase of mast cell number (MCN) is matched by a marked degranulation. No sex differences are found throughout the year. Manipulations of the photoperiod and temperature, either in winter or in summer, suggest that only the latter is responsible for the annual variations. Exposure to higher temperatures causes a decrease in the MCN in the winter frogs, while exposure of the summer frogs to low temperatures provokes the opposite effect. The pituitary gland also influences MCN. Hypophysectomy causes a decrease of MCN, with a return to normal following replacement therapy with homologous pars distalis homogenate. Among pituitary hormones, only ACTH mimics the effect of pars distalis homogenate. However, a possible link seems to exist between environmental (temperature) and hormonal (pituitary) factors, since hypophysectomy prevents the increase of MCN in the summer frogs exposed to low temperatures.

Animals↗

[Properties of sex steroid-binding protein from Xenopus laevis blood serum and detection of an estradiol-binding component in frog liver cytosol which differs from sex steroid-binding protein].

Binding and physico-chemical properties of sex steroid-binding protein (SBP) from blood serum and those of estrogen-binding components from liver cytosol of pubertal male and female species of clawed frog Xenopus laevis were studied. It was shown that SBP from both sex species of X. laevis specifically binds estradiol (E2) (Ka=5 . 10(6) M-1). Concentration of SBP binding sites for E2 is 7 . 10(-12) mole per mg of protein. Testosterone 5alpha-dihydrotestosterone and E2 effectively compete with [3H]-E2 for SBP binding sites. Hexestrol, progesterone and corticosterone are weak competitors; estrone and E2-17-hemisuccinate do not compete at all. The Strokes radius of SBP is 4.4 nm; sedimentation coefficient is 4.6S. Molecular weight of SBP is 88000; f/f0 is 1.5 SBP from male frog sera has been purified 8.6-fold with 13% yield. Gel-filtration of [3H]-E2 complexes with liver cytosol proteins shows that the livers of male and female frog X. laevis consol proteins shows that the livers of male and female frog X. laevis contain very low amounts of macromolecular component, which specifically binds E2; this component differs from serum SBP in size and in hormonal specificity. It is assumed that this component is a receptor for estrogens.

Animals↗

[Uptake of cyclic adenosine-3,5-monophosphate by cultured mammalian cells and frog muscles].

Uptake of 3H-cAMP by isolated frog sartorii muscles and cultured mouse 3T3 and 3T6 cells was studied. It was shown that after 1-2 hours of incubation the intracellular level of cAMP in frog muscles reached 10-20% of its concentration in the incubation medium. About 50% of intracellular radioactivity was represented by chromatographically pure cAMP which testifies to the insignificant cAMP metabolism rate. In the experiments with 3T3 and 3T6 cells the influence of possible admixtures and degradation products on 3H-cAMP uptake was revealed. To avoid these influences it is necessary to measure the uptake of cAMP in the presence of theophylline and with abundance of adenosine. In such experimental conditions the intracellular level of cAMP after 1 hour of incubation did not exceed 10% of its extracellular level, which is similar to values obtained on frog muscles. Permeability coefficient of cAMP for membrane of frog muscles and 3T3 and 3T6 cells was about 10(-9)-10(-8) cm X sec-1.

Adenosine↗

The properties of the extraocular muscles of the frog. II. Pharmacological properties of the isolated superior oblique and superior rectus muscles.

The pharmacological properties of the superior oblique and the superior rectus muscles of the frog's eye were investigated in comparison with those of a skeletal muscle (iliofibularis muscle) of the same animal. Acetylcholine causes sustained contractures of the extraocular muscles; this effect is increased by physostigmine and decreased or abolished by d-tubocurarine. Also the applications of succinylcholine, choline or caffeine are able to evoke contractures. There are no striking differences in pharmacological properties between extraocular and skeletal muscles of the frog. The time-course of the contractures and the sensitivity of the muscle preparations to the drugs which evoke contractures are identical in extraocular and iliofibularis muscles. In comparison with skeletal muscles there is no higher sensitivity of the extraocular muscles against curare-like drugs. The existence of adrenergic receptors could not be found neither in extraocular nor in skeletal muscles of the frog. It is concluded that in frogs no pharmacological differences exist between the muscle fibre types which compose the extraocular and the skeletal muscles.

Acetylcholine↗

Electron cytochemistry of peroxidase activity in neutrophils of the bone marrow of the frog Rana temporaria.

The bone marrow of the frog was studied during the spring and winter periods. During the winter period, one type of large granules in the neutrophils of the frog proved to be peroxidase-positive. The remaining, smaller granules are peroxidase-negative. In the spring, immature and mature neutrophils are encountered in the frog. The mature neutrophils of the frog in the spring and winter periods are analogous. The immature neutrophils contain peroxidase activity in the perinuclear space, the endoplasmic reticulum and some of the granules. Some of the peroxidase-containing granules are directly associated with the canals of the endoplasmic reticulum. In the immature neutrophils, during the formation of the peroxidase-containing granules, peroxidase-negative granules smaller in size are already present in the cytoplasm. Thus, the sequence of granule formation usual for mammals is apparently not observed in the neutrophils of certain amphibia. "Annular" mitochondria, encompassing part of the cytoplasm were detected in the neutrophils. These apparently represent a section through a spherical mitochondrion enveloping and isolating a certain zone of the cell. It is proposed that the mitochondria in this way, differing from the lysosomal mode, participate in the catabolism of the cytoplasm.

Animals↗

Extracellular space of frog skeletal muscle in vivo and in vitro: relation to proton magnetic resonance relaxation times.

1. The Na and Cl distribution spaces of freshly isolated frog muscles are 16.7 and 12.6%, respectively. These values increase to 25.6 and 23.3%, respectively, on incubation. 2. The extracellular components of both Na and Cl efflux curves are significantly smaller in freshly isolated muscles (approximately 12%) than in incubated muscles (approximately 18%). The fast exchanging A component of the extracellular space is increased more by incubation than the more slowly exchanging B component. 3. The proton magnetic resonance (p.m.r.) transverse relaxation curve for the water of freshly isolated frog muscles did not show the long, slowly relaxing tail present in curves from muscles incubated in Ringer solution. 4. When muscles were incubated in hypertonic solutions the p.m.r. transverse relaxation curves could be resolved into three components whose sizes were consistent with the components present in the sodium and chloride efflux curves. The non-exponentiality of the p.m.r. transverse relaxation curve therfore appears to arise from water in both the A and B extracellular compartments of muscle. 5. Efflux analysis indicated that the cellular Na content of both freshly isolated and incubated frog muscle is similar to that predicted by others (Lev, 1964; Armstrong & Lee, 1971; Lee & Armstrong, 1974) from measurements of intracellular Na ion activity using Na-sensitive micro-electrodes. The remainder of the tissue Na was found in the more rapidly exchanging extracellular compartments. The results of these experiments are inconsistent with the presence of a substantial fraction of bound Na in frog muscle. 6. These experiments show that muscle extracellular space is smaller in vivo than in vitro. Efflux analysis is suggested as the most accurate method of assessing extra-cellular components.

Animals↗

Distribution of 32P-labelled endotoxin in the frog.

Distribution and elimination of 32P-labelled endotoxin were studied in normal frogs and in frogs pretreated with a high dose of unlabelled endotoxin 24 hr previously. Like in other animals, the spleen and the liver of the frogs were found the most active R.E.S. organs. The alimentary tract too exhibited considerable R.E.S. activity. Pretreatment with unlabelled endotoxin resulted in a decrease of 32P-endotoxin uptake by the liver, the spleen and the alimentary tract. Evidence was obtained that the well-known effects of endotoxin on the R.E.S. of the mammals can be demonstrated in the frog as well.

Animals↗

[Super-long latent somato-sympathetic response in non-anesthesized decerebrate frogs].

Somato-sympathetic reflex responses were studied by recording the activity of the renal sympathetic efferents following excitation of sciatic nerve A-afferents in immobilized decerebrated frogs before and during viadril-induced anesthesia. Apart from A-response reported in anesthetized frogs and consisting of excitatory and inhibitory components, in non-anesthetized frogs reflex discharge with a latency over 2 sec was revealed. Unlike the former one, this response disappeared after intravenous injection of viadril. In the same frogs intravenous injection of viadril converted pressor reflexes in response to stimulation of sciatic nerve A-afferents into depressor ones. A-response with superlong latency is assumed to reflect the excitation of those central structures that are responsible for the development of pressor reflexes to somatic A-fiber stimulation. In this respect the described somato-sympathetic A-response seems to be analogous to the very late A-response in the mammals.

Anesthesia↗

Vascular dissemination of tumor cells in relation to temperature-dependent metastasis in frogs.

Metastasis is a temperature-related phenomenon in the North American leopard frog (Rana pipiens) affected with the Lucké renal adenocarcinoma. The frog is a poikilothermic vertebrate whose internal body temperature closely follows that of the environment, and tumor-bearing frogs living in a warm (28 degrees C) environment have a much higher incidence of metastasis (greater than 75%) than those in cold (4 degrees C) conditions (less than 6%). In this investigation it was found that Lucké tumor cells labeled with fluorescein isothiocyanate could be detected in frozen sections of all organs examined within 15 minutes, regardless of whether the hosts were adapted to a warm or a cold environment. Separate experiments involving inoculation of isotope-labeled tumor cells demonstrated that large numbers of cells arrived in the liver and other organs of animals kept at either end of the temperature range. These findings show that the infrequency of metastasis by Lucké adenocarcinomas in chilled frogs is not due to failure of dissemination of tumor cells consequent upon temperature-mediated changes in blood viscosity or flow patterns. Thus they establish the important baseline that the effects of temperature on metastasis in this vertebrate are exerted directly on the tumor cells and/or the internal environment of the host and that the system therefore provides new opportunities for probing the biology and biochemistry of tumor metastasis.

Adenocarcinoma↗

Importance of the visual cortex for postural stabilization: inferences from pigeon and frog data.

Optokinetically induced self-motion with its consequences for postural balance is based upon visual-vestibular convergence. It is a matter for speculation which visual pathways--subcortical accessory optic tract and/or cortical striate projection--convey optokinetic information to the central vestibular system. The functional significance of the visual cortex was tested by a behavioral approach in two animals, selected for their different evolutionary stage: frog (midbrain visual center); pigeon (primitive neocortical center). Lateral postural sway during optokinetic stimulation in roll served as a measurement for induced rollvection and apparent body tilt. Roll motion elicits a tonic 'compensatory' postural adjustment towards the direction of pattern motion in pigeon as in man but not in frog. From the lack of this reaction we infer that the frog does not perceive rollvection because it has no visual cortex. This agrees with the absence of visual-vestibular convergence in the frogs vestibular nuclei neurons as well as the absence of a nystagmus velocity storage in the brainstem. The animal experiments fit human data in hemianopic patients who also only experience rollvection when stimulated in the unaffected hemifield.

Animals↗

[Cytophotometric analysis of the cell population composition of the the preoptic area of the hypothalamus in the common frog].

DNA contents in squashed cells of the adult frog hypothalamic preoptic region (HPR) were measured using the Feulgen and UV cytophotometry techniques. The histone-DNA ratio in the cell nucleus was determined by means of a combined Feulgen-heparin-Alcian blue staining procedure. The nuclei of the vast majority of HPR cells have a diploid DNA content. However, in cells of this group the mean values of DNA amount and the distribution range were always higher than those in hepatocytes used as a diploid standard. Such a heterogeneity in DNA content in the diploid part of HPR cell population could apparently suggest some differences in the nuclear chromatin arrangement to be always higher in spring before the frog spawning, and it seems to be characteristic of this type of cells. About 1 per cent of cells with hyperdiploid surplus of DNA (H2c cells) as well as of tetraploid cells (4c and 2c X 2 cells) is found in HPR in frogs sacrificed both in winter and in summer. The quota of these cells has no reference either to the frog's age or to the annual cycle. The fact that the mean DNA values in H2c and 4c cells are much higher than in the standard cannot be explained by the presence of different amounts of nuclear proteins only. It is suggested that at least some part of the highest DNA values may be due to an actual extra DNA synthesis in a small constantly existing pool of HPR cell population.

Animals↗

Anatomical symmetry in the hind limbs and spinal motoneurons in frogs.

The weight of muscles and bones of the right and left hind limbs has been studied in 60 frogs. In the first ten frogs studied, the number and size of motoneurons of the seventh, eighth and ninth lumbar spinal segments (which give origin to the sciatic nerve) were studied on both sides. This was done to ascertain the relationship between motoneuron size and limb dominance. No statistically significant difference was observed in the weights of the right and left hind limbs indicating that frogs do not show one-sided hind limb dominance. This result stands in marked contrast to a previous report of asymmetry in bone weight in frogs. Similarly no significant difference was observed in the numerical distribution of neurons and their size between the two sides of the spinal cord.

Animals↗