Intracelomic use of tricaine methanesulfonate for anesthesia of bullfrogs (Rana catesbeiana) and leopard frogs (Rana pipiens)

The use of intracelomic injection of dissolved tricaine methanesulfonate (MS-222) as an anesthetic agent in two anuran species was studied. Intracelomic MS-222, at dosages of 100, 250, and 400 mg/kg, rapidly induced tranquilization or anesthesia. Effects were less pronounced or nonexistent at the 50 mg/kg dosage. Depth and duration of anesthesia were dosage related. At the 100, 250, and 400 mg/kg dosages, Rana pipiens attained a greater depth of anesthesia and remained anesthetized for a significantly greater duration than did R. catesbeiana. Dosages of between 250–400 mg/kg reliably induced deep anesthesia without mortality in bullfrogs. Dosages of less than 250 mg/kg are recommended for leopard frogs, since variable mortality was noted with higher dosages. Solubilized tricaine methanesulfonate did not cause gross or histopathological lesions to celomic tissues. Tricaine methanesulfonate injected intracelomically can provide rapid, efficient anesthesia in some anuran species. However, due to the observed intra- and interspecies variation in effect, it should be used cautiously, especially in unfamiliar species. © 1992 Wiley-Liss Inc.     

Vitamin A enhances forelimb regeneration in juvenile leopard frogs Rana pipiens

The effect of retinoids on forelimb regeneration in the leopard frog, Rana pipiens, was examined by the topical application of a concentrated solution of vitamin A palmitate to the amputated left limb stumps of 30 froglets. This treatment resulted in the enhancement of regeneration; regenerative outgrowths of experimental frogs were more frequent, larger in size, and had improved morphology compared to their untreated control counterparts. We conclude that vitamin A palmitate, topically administered in concentrated dosages, exerts a positive effect on the regenerative capacity of Rana pipiens.     

Large intestine bacterial flora of nonhibernating and hibernating leopard frogs (Rana pipiens).

The bacteria in the large intestines of 10 northern leopard frogs (Rana pipiens) were enumerated and partially characterized. Four nonhibernating frogs were collected in the summer, four hibernating frogs were collected in the winter, and two frogs just emerged from hibernation were collected in the spring. All frogs had about 10(10) bacteria per g (wet weight) of intestinal contents and about 10(9) bacteria per g (wet weight) of mucosal scraping, although the counts from the winter frogs were slightly less than those from the other two groups of frogs. Another group of 14 summer frogs, after treatment to induce hibernation, showed a drop in bacterial counts accompanied by a change in the composition of the flora. In most frogs, Bacteroides was the dominant organism. Other bacteria repeatedly isolated at high dilutions were strict anaerobes, including butyrigenic and acetogenic helically coiled bacteria; fusobacteria; and acetogenic, small, gram-positive bacilli. These data indicate that the intestinal flora of frogs is similar to that of mammals and birds and that this flora can be maintained at temperatures close to freezing.     

Possible mechanisms responsible for the reduced intestinal flora in hibernating leopard frogs (Rana pipiens)

Mechanisms and factors that normally control the large intestinal flora were investigated to determine whether changes in these parameters could account for the decreased bacterial concentration and facultative nature of the flora found in hibernating frogs. It appeared that low temperatures and limited nutrients were the main factors responsible for the decrease in the bacterial concentration and may also have been responsible for the increase in the proportions of facultative organisms, since no change in the redox potential was seen. The hibernating frogs were extremely sluggish in the removal of India ink particles from the circulatory system by the Kupffer cells of the liver compared with nonhibernating frogs. They were unable to mount an antibody response to bovine serum albumin, but their serum did exhibit killing of Pseudomonas paucimobilis, suggesting opsonization by preformed antibody and complement. The role of these host factors in protecting the hibernating frog against this indigenous flora is discussed.     

The ebb and flow of antimicrobial skin peptides defends northern leopard frogs (Rana pipiens) against chytridiomycosis

Many amphibian species are threatened with extinction by the emerging infectious disease, chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis. This unprecedented global crisis threatens to reduce the biodiversity of the entire amphibian class. The fungus invades the skin and impairs the uptake and retention of essential ions leading to cardiac arrest. Antimicrobial peptides (AMPs) secreted into the mucus of some amphibians are thought to be an important defense against chytridiomycosis. However, little is known about the quantities of AMPs secreted under natural conditions, whether they are sufficient to protect against this pathogen, and how they interact with commensal microbes. To understand how defensive peptides and skin microbes may interact, it is essential to know the precise quantities of AMPs present under natural conditions. Using matrix‐assisted laser desorption time‐of‐flight mass spectrometry and growth inhibition assays, we show that northern leopard frogs (Rana pipiens) at rest constitutively release low amounts of AMPs that inhibit B. dendrobatidis in vitro, and AMP defenses are elevated following a simulated predator attack. Using a synthetic peptide analogue of brevinin‐1Pb as an external control, we quantified the amounts of four previously described AMPs (brevinin 1Pa, brevinin‐1Pb, brevinin‐1Pd, and ranatuerin‐2P) at several time points after secretion. Once secreted onto the skin, the peptides are most active for 15 min, and small quantities persist for at least 2 h. Taken together, our data suggest that small amounts of AMPs are rapidly available and quite stable on the skin of R. pipiens. They are effective inhibitors of B. dendrobatidis at these low constitutive concentrations but degrade within 2 h, protecting the integrity of the skin and commensal bacteria.     

Response of intestinal flora of laboratory-reared leopard frogs (Rana pipiens) to cold and fasting.

The bacterial flora of the large intestine was examined in 35 laboratory-reared leopard frogs (Rana pipiens) subjected to one of the following four treatments: (i) normal feeding at 21 degrees C (10 frogs); (ii) fasting for 2 weeks at 21 degrees C (8 frogs); (iii) chilling for 1 week at 4 degrees C (9 frogs); and (iv) simulated hibernation for 3 weeks at 4 degrees C (8 frogs). Bacteria from the intestinal contents and mucosa were counted microscopically and by colony counting after strictly anaerobic culturing. The predominant bacteria were isolated and partially characterized. Fasting for 2 weeks produced no significant changes in total counts or in the types of bacteria cultured. Chilling, whether rapid or in the course of simulated hibernation, was associated with a decrease in the numbers and variety of bacteria. Thus it appears that the lowering of temperature rather than the absence of food is the important factor in the reduction of bacterial flora seen in hibernating frogs. However, the bacteria showed some adaptation to the low temperature, as the longer the host had been at 4 degrees C, the higher the proportion of bacteria which could grow when cultured at that temperature.     

A study on hearing in frogs (Rana pipiens and Rana clamitans)

Zusammenfassung Die relative Reizschwellkurve für die Schallempfindung bei den Fröschen Rana clamitans und R. pipiens wurde in dem Frequenzbereich von 30–15000 Hz bestimmt. Die Kurve ist dieselbe für beide Geschlechter. Versuche zeigten, daß Schälle entweder inhibierend oder stimulierend auf die Atmungsbewegungen wirken können. Diese Wirkung wird nicht von der Frequenz oder der Intensität der Schälle bestimmt, sondern von dem physiologischen Zustand des Tieres.Die Latenzzeit zwischen Reiz und Effekt ist unabhängig von der Tonfrequenz, wird aber von den Intervallen zwischen aufeinanderfolgenden Reizen beeinflußt; sie wird kürzer, wenn die Intervalle verlängert werden.Dressurversuche mit 18 Tieren, unter Anwendung von Strafung durch elektrische Schläge und einem Dressurton von 300 Hz, ergaben positive Erfolge bei zwei Individuen von R. clamitans.

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Contribution No. 67 of the Department of Biology, Hamilton College, McMaster University, Hamilton, Ontario.     

Classification and distribution of large intestinal bacteria in nonhibernating and hibernating leopard frogs (Rana pipiens).

The large intestinal flora of the leopard frog, Rana pipiens, was examined to determine whether differences existed between the nonhibernating and hibernating states of the animal and to determine the relative concentrations and proportions of potential frog pathogens. Hibernators had a logarithmic decrease of bacteria per milligram of intestine averaging one, and significantly greater proportions of facultative bacteria and psychrophiles relative to nonhibernators. The predominant anaerobic bacteria were gram-positive Clostridium species and gram-negative Bacteroides and Fusobacterium species. The predominant facultative bacteria were enterobacteria in nonhibernators but Pseudomonas species in hibernators. Many species of Pseudomonas are pathogenic for frogs, and thus the intestinal flora in hibernators may be a potential source of infectious disease.     

Possible mechanisms responsible for the reduced intestinal flora in hibernating leopard frogs (Rana pipiens).

Mechanisms and factors that normally control the large intestinal flora were investigated to determine whether changes in these parameters could account for the decreased bacterial concentration and facultative nature of the flora found in hibernating frogs. It appeared that low temperatures and limited nutrients were the main factors responsible for the decrease in the bacterial concentration and may also have been responsible for the increase in the proportions of facultative organisms, since no change in the redox potential was seen. The hibernating frogs were extremely sluggish in the removal of India ink particles from the circulatory system by the Kupffer cells of the liver compared with nonhibernating frogs. They were unable to mount an antibody response to bovine serum albumin, but their serum did exhibit killing of Pseudomonas paucimobilis, suggesting opsonization by preformed antibody and complement. The role of these host factors in protecting the hibernating frog against this indigenous flora is discussed.     

The germfree leopard frog (Rana pipiens): preliminary report.

Eggs and sperm were aseptically collected from leopard frogs (Rana pipiens) and passed into sterilized flexible film isolators. Egg fertilization was accomplished, and germfree tadpoles were produced. The germfree tadpoles were active and free-swimming for 3 weeks, but only 6.2% survived to 4 weeks of age. None survived after the 4th week.     

Phosphate metabolism during muscular contraction in starved frogs (Rana catesbeiana)

• 1.1. The muscle tension and the state of high-energy phosphate metabolism during contraction of the sartorius muscle in frogs (Rana catesbeiana) starved for 1–5 months was studied by in vivo³¹P-NMR spectrometry.
• 2.2. Muscle tension began to decrease after 2-month starvation compared with the control group and decreased to about one-third of the control value after a 5-month starvation.
• 3.3. Muscle contraction induced by electrical stimulation or the use of anaerobic perfusion fluid did not decrease the concentration of creatine phosphate (PCr) or β-ATP, and only negligibly changed the PCr/Pi ratio from starvation.
• 4.4. These results suggest a decrease in creatine kinase activity in the muscle of starved frogs.

     

Mate calling induced by electrical stimulation in freely moving leopard frogs, Rana pipiens.

Electrodes were implanted chronically into the preoptic areas of normal or castrated male frogs, Rana pipiens, and monopolar monophasic stimulating currents (100 Hz, 0.5-msec duration, and mainly 50–200 μA in intensity) were delivered through the implanted electrodes in freely moving frogs. When the electrodes were placed in the rostral part of the preoptic nucleus, mating calls (mainly trills and sometimes chuckles) were inducible. Fifty microamperes was generally an effective stimulus intensity for induction of calls, and 20 μA was the minimum effective stimulus intensity observed. There was no difference in the threshold to induce calls between pituitary-treated intact and castrated frogs.     

Phylogenetic relationships of leopard frogs (Rana pipiens complex) from an isolated coastal mountain range in southern Sonora, Mexico

Mitochondrial DNA sequence data from the control region and 12S rRNA in leopard frogs from the Sierra El Aguaje of southern Sonora, Mexico, together with GenBank sequences, were used to infer taxonomic identity and provide phylogenetic hypotheses for relationships with other members of the Rana pipiens complex. We show that frogs from the Sierra El Aguaje belong to the Rana berlandieri subgroup, or Scurrilirana clade, of the R. pipiens group, and are most closely related to Rana magnaocularis from Nayarit, Mexico. We also provide further evidence that Rana magnaocularis and R. yavapaiensis are close relatives.     

Hind limb malformations in free-living northern leopard frogs (Rana pipiens) from Maine, Minnesota, and Vermont suggest multiple etiologies

BACKGROUND: Reports of malformed frogs have increased throughout the North American continent in recent years. Most of the observed malformations have involved the hind limbs. The goal of this study was to accurately characterize the hind limb malformations in wild frogs as an important step toward understanding the possible etiologies. METHODS: During 1997 and 1998, 182 recently metamorphosed northern leopard frogs (Rana pipiens) were collected from Minnesota, Vermont, and Maine. Malformed hind limbs were present in 157 (86%) of these frogs, which underwent necropsy and radiographic evaluation at the National Wildlife Health Center. These malformations are described in detail and classified into four major categories: (1) no limb (amelia); (2) multiple limbs or limb elements (polymelia, polydactyly, polyphalangy); (3) reduced limb segments or elements (phocomelia, ectromelia, ectrodactyly, and brachydactyly; and (4) distally complete but malformed limb (bone rotations, bridging, skin webbing, and micromelia). RESULTS: Amelia and reduced segments and/or elements were the most common finding. Frogs with bilateral hind limb malformations were not common, and in only eight of these 22 frogs were the malformations symmetrical. Malformations of a given type tended to occur in frogs collected from the same site, but the types of malformations varied widely among all three states, and between study sites within Minnesota. CONCLUSIONS: Clustering of malformation type suggests that developmental events may produce a variety of phenotypes depending on the timing, sequence, and severity of the environmental insult. Hind limb malformations in free-living frogs transcend current mechanistic explanations of tetrapod limb development.     

Cytopathological observations and epizootiology of frog erythrocytic virus in bullfrogs (Rana catesbeiana).

A 5 yr survey (1985 to 1989) revealed that the prevalence of frog erythrocytic virus (FEV) was significantly higher in juveniles than in adult bullfrogs (Rana catesbeiana) in Algonquin Park, Ontario (Canada). The prevalence of infection in juveniles declined during May and June and increased in early August, persisting until late September. Tadpoles were not infected naturally with FEV, but were susceptible to experimental infection. Experimental data indicate that FEV is neither waterborne, nor transmitted by the oral route, nor by the leech Desserobdella picta. The virus may be transmitted mechanically by the mosquito Culex territans, or the midge Forcipomyia (Lasiohelea) fairfaxensis. Nine percent recapture of marked uninfected frogs versus 4% recapture of infected animals suggests that FEV contributes to mortality of juvenile bullfrogs. Infected erythrocytes were transformed from ellipsoidal to spheroidal cells, some of which contained flattened elongate, trapezoidal inclusions. There was no evidence of denatured hemoglobin or significant change in the hemoglobin content of infected cells. Anemia recorded in heavily infected animals was not attributable to an increased osmotic fragility of infected erythrocytes.     

Enterobacteriaceae and Aeromonas hydrophila in Minnesota frogs and tadpoles (Rana pipiens)

In 222 Rana pipiens frogs and 34 tadpoles captured in and near Minnesota, Aeromonas hydrophila and 29 species of Enterobacteriaceae, including yersinia enterocolitica and Salmonella arizonae, were isolated from intestines. The prevalence of members of the family Enterobacteriaceae was lowest in frogs captured in early spring and highest in frogs captured in late summer.     

Immunocytochemistry of luteinizing hormone-releasing hormone in brains of bullfrogs (Rana catesbeiana) during spontaneous metamorphosis

The distribution of immunoreactive luteinizing hormone-releasing hormone (ir-LH-RH) in brains of bullfrogs (Rana catesbeiana) during spontaneous metamorphosis has been studied by combination of an unlabeled antibody enzyme immunocytochemical technique and an adjacent serial section approach. In prometamorphic tadpoles, immunocytochemical staining for ir-LH-RH was absent from the brain, including a structurally simple median eminence (ME) and perikarya in the anterior preoptic area (aPOA). In metamorphic tadpoles, speckled patches of immunostaining occurred over the outer layer of a modestly developed ME; coincident faint staining of a small number of medial, unpaired cell bodies was localized in the aPOA. In newly metamorphosed juvenile frogs, more diffuse and intense staining of the outer layer of the ME accompanied increased morphological differentiation of this neurohemal area; immunoreactive perikarya again were found in the aPOA, but an increased number of neurons exhibited comparatively greater (moderate) immunostaining. Changes in the quality of immunostaining and in the numbers of cells stained, therefore, were coincident with metamorphic development. Concomitant alterations of ir-LH-RH immunostaining and progressive structural development of the ME suggest a coordinated differentiation of brain neuroendocrine systems during metamorphosis of the bullfrog tadpole.