Description of a new octoploid frog species (Anura: Pipidae: Xenopus) from the Democratic Republic of the Congo, with a discussion of the biogeography of African clawed frogs in the Albertine Rift.

We describe a new octoploid species of African clawed frog (Xenopus) from the Lendu Plateau in the northern Albertine Rift of eastern Democratic Republic of the Congo. This species is the sister taxon of Xenopus vestitus (another octoploid), but is distinguished by a unique morphology, vocalization and molecular divergence in mitochondrial and autosomal DNA. Using a comprehensive genetic sample, we provide new information on the species ranges and intra-specific diversity of African clawed frogs from the Albertine Rift, including the details of a small range extension for the critically endangered Xenopus itombwensis and previously uncharacterized variation in Xenopus laevis. We also detail a new method for generating cytogenetic preparations in the field that can be stored at room temperature for up to 3 weeks. While extending our understanding of the extant diversity in the Albertine Rift, this new species highlights components of species diversity in ancestral African clawed frogs that are not represented by known extant descendants.

Cisticercose em animais abatidos em Sabáudia, Estado do Paraná / Cysticercosis in animals of Sabáudia, Paraná State

The aim of this work was to verify the occurrence of bovine and swine cysticercosis in animals slaughtered at the Abatedouro Municipal de Sabáudia, Paraná State. The number of daily-abated animals, the number of animals with cysticercus and the number and location of the cysticercus in the animals in a period of twelve months were collected. From 1046 swines slaughtered none showed cysticercus, but 36/out of 389 bovines (9.3 percent) were contaminated. Head was the most parasited region, followed by heart, tongue and liver. The absence of contamination observed in swines and the low contamination of the bovines reinforce the efficiency of the control measures accomplished in Paraná State.

Estrogen and laryngeal synaptic strength in Xenopus laevis: opposite effects of acute and chronic exposure.

Synaptic transmission at the vocal synapse, the laryngeal neuromuscular junction, of Xenopus laevis has been shown to be regulated by long-term changes in circulating estrogen. In females, high levels of circulating estrogen also accompany gonadotropin-induced ovulation and oviposition and the switch from sexually unreceptive to receptive states, including changes in vocal behaviors (ticking to rapping). Here we examine the effects of gonadotropin injection on laryngeal synaptic strength and call type. Gonadotropin acutely reduced quantal content values of laryngeal synapses in intact, adult females; the lowest values were attained by 12 h post-injection. Estrogen and progesterone levels increased following human chorionic gonadotropin (hCG) injection; the time course was similar to, but negatively correlated with, changes in synaptic strength. In ovariectomized frogs, exogenous estrogen, but not progesterone or hCG, mimicked the acute effects of hCG in weakening laryngeal synapses of intact frogs. hCG injection suppressed ticking and sometimes induced rapping. Females could tick with either strong or weakened laryngeal synapses while rapping was only produced during the weakening action of hCG. The normally strong synapses of females may enable vocal production even when laryngeal synapses are weakened by hormones that induce ovulation. In contrast to the acute effect of estrogen on weakening laryngeal synapses, juveniles required more than 2 weeks of estrogen treatment to strengthen laryngeal synapses while at least 4 weeks postovariectomy were required to weaken synapses in adult females. We conclude that acute (hours) increases in circulating levels of estrogen weaken synapses while chronic (weeks) increases strengthen laryngeal synapses.

Attaining and maintaining strong vocal synapses in female Xenopus laevis.

Synaptic efficacy at the laryngeal neuromuscular synapse differs markedly in adult male and female Xenopus laevis. Here, we examined the relation between circulating estrogen and synapse strength in developing and adult female frogs. Circulating estrogen levels in males and females during juvenile and adult stages were measured using radioimmunoassays. Synaptic strength was determined by quantal analysis in isolated female larynges. In males, estrogen levels are low (<40 pg/mL) throughout development. In females, estrogen levels are similar to those in males until 9 months after metamorphosis is complete and then increase throughout development. Female laryngeal synapses have low quantal contents until 24 months; quantal content increases significantly between 24 and 26 months, and high quantal contents are maintained thereafter. Measures of reproductive maturation, ovary, and oviduct weights, are strongly and positively correlated with estrogen level in 16- to 26-month females, while oocyte maturation is age dependent. Estrogen level and quantal content are not well correlated in these females. Ovariectomy at 24 months prevents the expected increase in quantal content and ovariectomy at 28 months results in a decrease in quantal content. Thus, the sex difference in efficacy of the laryngeal synapse develops under the influence of the ovary and requires the ovary for maintenance of strong synapses in adulthood. While the influence of the ovary is most likely due to estrogen secretion, the pattern of estrogen secretion required for maturation of the synapse in females is not known.

Rapping, a female receptive call, initiates male-female duets in the South African clawed frog.

Finding a sexually receptive partner of the opposite sex is a challenge; one solution is to advertise. That advertising is usually the province of males has shaped scenarios for sexual selection, especially the ardent active male courting the passive but choosy female. Herein we consider an unusual case in which constraints on reproduction may have led to fertility advertisement by female frogs. When oviposition is imminent, female South African clawed frogs swim to an advertising male and produce an aphrodisiac call, rapping, that stimulates both male vocalization and approach. Males respond to rapping with a distinctive answer call. The rapping-answer interaction thus forms a duet between partners of a receptive pair.

Facilitation at the sexually differentiated laryngeal synapse of Xenopus laevis.

Under physiological conditions, the laryngeal synapse of male Xenopus laevis exhibits marked facilitation during repetitive nerve stimulation. The male laryngeal synapse is weak and requires facilitation to produce muscle action potentials and ultimately sound. The female laryngeal synapse is strong: muscle contractions are produced to single nerve stimuli. We sought to determine if laryngeal synapses of males and females also differ in their ability to facilitate. To measure facilitation, laryngeal muscle action potentials were suppressed either postsynaptically by bathing the preparation in saline containing curare or presynaptically by bathing the preparation in reduced calcium/elevated magnesium saline. Facilitation of postsynaptic potential amplitude or quantal content in response to paired pulses was measured in male and female larynges: there is no sex difference in paired pulse facilitation. Facilitation in response to trains of stimuli, in curare-blocked preparations, increased and reached plateau values more rapidly in females than in males, although the facilitation between the last and first pulses in the train was the same in the sexes. Thus, the sexually differentiated behavior of this synapse is controlled more by a sex difference in synaptic strength than by a sex difference in the ability to facilitate.

Sexual differentiation and hormonal regulation of the laryngeal synapse in Xenopus laevis.

In Xenopus laevis frogs, sex differences in adult laryngeal synapses contribute to sex differences in vocal behavior. This study explores the development of sex differences in types of neuromuscular synapses and the development and hormone regulation of sex differences in transmitter release. Synapses in the juvenile larynx have characteristics not found in adults: juvenile muscle fibers can produce subthreshold or suprathreshold potentials in response to the same strength of nerve stimulation and can also produce multiple spikes to a single nerve stimulus. Juvenile laryngeal muscle also contains the same synapse types (I, II, and III) as are found in adult laryngeal muscle. The distribution of laryngeal synapse types in juveniles is less sexually dimorphic than the distribution in adults. Analysis of quantal content indicates that laryngeal synapses characteristically release low amounts of transmitter prior to sexual differentiation. Quantal content values from male and female juveniles are similar to values for adult males and are lower than values for adult females. When juveniles are gonadectomized and treated with exogenous estrogen, quantal content values increase significantly, suggesting that this hormone may increase transmitter release at laryngeal synapses during development. Gonadectomy alone does not affect quantal content of laryngeal synapses in either sex. Androgen treatment decreases quantal content in juvenile females but not males; the effect is opposite to and smaller than that of estrogen. Thus, muscle fiber responses to nerve stimulation and transmitter release are not sexually dimorphic in juvenile larynges. Transmitter release is strengthened, or feminized, by the administration of estradiol, an ovarian steroid hormone.

A sex difference in synaptic efficacy at the laryngeal neuromuscular junction of Xenopus laevis.

Under physiological conditions, the response of Xenopus laevis laryngeal muscle fibers to nerve stimulation is sexually differentiated; subthreshold potentials are common in males and rare in females. This sex difference in muscle fiber response is correlated with sex differences in vocal behavior. Quantal analyses at male and female laryngeal synapses were performed to determine if there is a sex difference in synaptic strength. Quantal content at laryngeal synapses is significantly higher in females than in males. Values for quantal content in males can be increased by raising extracellular calcium concentration. There is no sex difference in miniature endplate potential amplitude suggesting that ACh receptor number or properties are not different in the sexes. Sex difference in synaptic strength thus appear presynaptic in origin; transmitter release is less in males. Ultrastructural analyses of the laryngeal motor terminal indicate that there is no sex difference in the length of active zones or in the number of channels per length of active zone. Thus, ultrastructural characteristics of the laryngeal motor terminal do not account for the pronounced sex difference in quantal content.

The roles of sex, innervation, and androgen in laryngeal muscle of Xenopus laevis.

The relative contributions of innervation and androgen to three muscle fiber properties--twitch type, size, and number--were examined in the sexually dimorphic, androgen-sensitive laryngeal muscle of Xenopus laevis. In adults, the muscle contains all fast-twitch fibers in males and fast- and slow-twitch fibers in females; laryngeal muscle fibers are larger and more numerous in males than in females. Juvenile larynges are female-like in both sexes; male laryngeal muscle is subsequently masculinized by androgen secretion during postmetamorphic development. Because both laryngeal motor neurons and muscle fibers are androgen sensitive during masculinization, we examined the role of the nerve in androgen-regulated muscle fiber development. Laryngeal muscle of male and female juvenile frogs was unilaterally denervated, and effects on muscle fiber type, size, and number were examined 4 weeks later. Half of the frogs received a dihydrotestosterone pellet at the time of denervation. Androgen treatment converts laryngeal muscle from mixed slow and fast to all fast twitch in both innervated and denervated muscle. Thus, the nerve is not required for androgen-regulated fiber type expression in either sex. Denervation produces muscle fiber atrophy and androgen treatment induces muscle fiber hypertrophy in male and female larynx. Nerve and hormone effects are independent and additive; fiber size in androgen-treated denervated muscle is greater than in untreated innervated muscle, and fiber size in androgen-treated denervated muscle is smaller than in androgen-treated innervated muscle. There is no sex difference in the effects of innervation or androgen on fiber size. Denervation causes laryngeal muscle fiber loss in males but not in females. Androgen treatment protects male laryngeal muscle from denervation-induced fiber loss and causes fiber addition in innervated female laryngeal muscle. We conclude that there is a sexually dimorphic interaction between innervation and androgen in control of laryngeal muscle fiber number.

Sexually dimorphic expression of a laryngeal-specific, androgen-regulated myosin heavy chain gene during Xenopus laevis development.

Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue.

Development of functional sex differences in the larynx of Xenopus laevis.

Three laryngeal properties associated with the production of masculine song--laryngeal muscle tension, fiber twitch type, and fiber recruitment--are markedly sexually dimorphic in adult Xenopus laevis frogs. To elucidate the pattern of sexual differentiation, tension and fiber recruitment in male and female larynges and fiber twitch type in male larynges were examined throughout postmetamorphic development. Masculinization of male laryngeal properties begins early in postmetamorphic development and continues until adulthood. In contrast, tension and fiber recruitment in females do not change after the end of metamorphosis. Laryngeal muscle tension and fiber type are gradually and progressively masculinized; the temporal pattern of masculinization is very similar for these properties. Fiber recruitment, on the other hand, appears to masculinize in a stepwise manner. Masculinization of all three properties is highly correlated with larynx weight in males. We have used this relation to divide postmetamorphic development into seven stages associated with key events in sexual differentiation. This staging scheme provides an important experimental tool for studying the hormonal regulation of sexual differentiation, the subject of the accompanying paper.

Temporal constraints on androgen directed laryngeal masculinization in Xenopus laevis.

Temporal constraints on androgen regulated masculinization of three sexually dimorphic laryngeal properties--tension, fiber type, and fiber recruitment--were examined in Xenopus laevis frogs. Endocrine state was manipulated at PM0 when the larynx is similar in males and females, at PM2 when the larynx begins sexual differentiation, and at PM6 when sexual differentiation is complete. Removing the testes in developing males (PM0 or PM2) completely arrests laryngeal masculinization. Masculinization resumes when testosterone is replaced later in development (PM2 or PM6, respectively). Thus, testicular secretions, in particular androgens, are required for laryngeal masculinization. The ability of androgens to masculinize tension, fiber type, and fiber recruitment in developing and adult larynges was also determined. Five weeks of testosterone treatment in PM0 or PM2 males and females completely masculinizes laryngeal tension and fiber type, but only partially masculinizes fiber recruitment. However, fiber recruitment can be fully masculinized in PM6 males castrated at PM2. We conclude that androgen induced masculinization of tension and fiber type are not temporally constrained but that androgen induced masculinization of fiber recruitment is. Prolonged androgen treatment can override the temporal constraints on masculinization of the larynx. Testosterone treatment for more than 6 months fully masculinizes fiber recruitment in developing (PM0 or PM2) females. In addition, prolonged treatment (greater than 9 months) completely masculinizes tension, fiber type, and fiber recruitment in adult females; these properties were not fully masculinized by shorter (1-3 months) treatments in adult females. Testosterone induced masculinization in females is maintained for up to 8 months following testosterone removal; thus androgen effects are long lasting and possibly permanent.

Hormone-sensitive stages in the sexual differentiation of laryngeal muscle fiber number in Xenopus laevis.

The number of muscle fibers in the vocal organ of the adult male African clawed frog, Xenopus laevis, exceeds that of adult females. This sex difference is the result of rapid fiber addition in males between the end of metamorphosis, post-metamorphic stage 0 (PM0) and PM2. At PM0, male and female frogs have similar numbers of laryngeal muscle fibers. Males then add more muscle fibers than females and achieve an adult value that is 1.7 times the female number. Males castrated at PM0 have the same fiber number as females. Ovariectomy at PM0 does not alter muscle fiber addition in females. Gonadectomy at PM2 has no effect on fiber addition in either sex. Females attain masculine muscle fiber number if their ovaries are replaced with a testis at metamorphosis. Exogenous testosterone treatment at PM0 significantly increases fiber number in females but not in males. Exogenous testosterone given at PM2 has no effect on fiber number in females but decreases fiber number in males. We conclude that the testes are necessary for the marked addition of laryngeal muscle fibers seen in male X. laevis between PM0 and PM2. The masculine pattern of muscle fiber addition can be induced in females provided with a testis. Androgen secretion from the testes most probably accounts for masculinization of laryngeal muscle fiber number. After PM2, androgens are no longer necessary for muscle fiber addition and cannot increase fiber number in females.

Electrophysiology and dye-coupling are sexually dimorphic characteristics of individual laryngeal muscle fibers in Xenopus laevis.

Sex differences at the laryngeal neuromuscular junction of Xenopus laevis were examined by recording intracellularly from muscle fibers in response to nerve stimulation. Male laryngeal muscle contains 2 physiologically distinct fiber types. Type I fibers generate postsynaptic potentials in response to low-magnitude stimulus pulses and action potentials in response to higher-magnitude stimulus pulses. Type II muscle fibers require repetitive stimulation for action potential production, probably because of facilitation. Subthreshold events in type I and II fibers suggest that these neuromuscular synapses have low safety factor junctions. Female laryngeal muscle contains one fiber type (III), which is physiologically distinct from those found in the male. Type III fibers produce an action potential in response to a single-stimulus pulse of suprathreshold voltage delivered to the laryngeal nerve; subthreshold events were not observed. Iontophoretic injection of Lucifer yellow into a single female muscle fiber resulted in as many as 43 labeled fibers. In males, only one fiber was labeled. Dye-coupling was not observed in adult females treated with the androgenic steroid hormone, testosterone. We have previously reported that laryngeal muscle fibers are recruited throughout a stimulus train presented to the laryngeal nerve in males, but are not recruited in females (Tobias and Kelly, 1987). Sex differences in the frequency of electrophysiological fiber types described here may account for sex differences in fiber recruitment. Synchronous activity of dye-coupled fibers may increase the effectiveness of muscle contraction in females.

Vocalizations by a sexually dimorphic isolated larynx: peripheral constraints on behavioral expression.

The clawed frog Xenopus laevis uses sexually dimorphic vocalizations, mate calling and ticking, to advertise reproductive state. The basic unit of vocalization is a brief click, produced by the movement of cartilagenous disks located within the larynx. The rate of click production in the male-specific mate call (71 Hz) is an order of magnitude faster than the rate of click production in female typical ticking (6 Hz). To determine if vocalization rate is constrained by the periphery, male and female larynges were isolated and response of the muscles to nerve stimulation was studied. Laryngeal muscle response is markedly dimorphic in the 2 sexes, both in the amplitude potentiation of electromyograms and in the rate at which discrete tension transients can be produced. At 6 Hz (ticking), both sexes generate discrete tension transients in response to each stimulus pulse. In response to nerve stimulation at 71 Hz (mate calling), male laryngeal muscle generates discrete tension transients while female laryngeal muscle does not. Since expression of sex-specific vocalizations is regulated by androgenic hormones, responses of laryngeal muscle to nerve stimulation in androgen-treated adult females and castrated adult males were also examined. The responses of laryngeal muscle from castrated and intact males are similar. Androgen-treated female larynx is partially masculinized but does not produce tension transients at the mate call rate. These physiological results are in close agreement with behavioral observations. Sounds produced by the isolated larynx were nearly identical in spectral properties to those produced by an intact male. We determined that the production of a discrete tension transient is prerequisite to click production. Thus, one reason females do not mate call, even when treated with androgens, is that female laryngeal muscle cannot produce discrete tension transients at a rapid rate.

Origin and identification of fibers in the cranial nerve IX-X complex of Xenopus laevis: Lucifer Yellow backfills in vitro.

The central projections of individual components of the IX-X nerve complex in the South African clawed frog, Xenopus laevis, were mapped by dye diffusion with Lucifer Yellow in an isolated brain preparation. The method reliably revealed fiber tracts, termination zones, and detailed cell morphology. In addition, motor neurons could be doubly labelled by retrograde transport of horseradish peroxidase from muscle targets followed by backfilling the appropriate nerves with Lucifer Yellow. The most anterior root associated with the nerve IX-X complex, root 1, is composed of lateral line afferents that terminate in the medial medulla. Root 2 contains sensory fibers that terminate in the nucleus tractus solitarii and axons of lateral line efferent neurons. Root 3 is composed of sensory and motor fibers, including a major somatosensory component that terminates in posterior medulla and anterior spinal cord, and axons from cranial nerve nucleus IX-X. The most posterior root of the IX-X nerve complex, root 4, contains axons of laryngeal motor neurons and of general visceral efferent neurons.

Flight Activity Initiated via Giant Interneurons of the Cockroach: Evidence for Bifunctional Trigger Interneurons.

Activity in dorsal giant interneurons of the cockroach initiates flight movements if leg contact with a substrate is prevented. The same interneurons initiate activity associated with running when leg contact is maintained. Thus, which one of two completely different behaviors the giant interneurons evoke depends on the presence or absence of leg contact.