Two novel antimicrobial peptides from skin secretions of the frog,Rana nigrovittata

Two novel antimicrobial peptides with similarity to brevinin‐2 family are purified and characterized from the skin secretions of the frog, Rana nigrovittata. Their amino acid sequences were determined as GAFGNFLKGVAKKAGLKILSIAQCKLSGTC (brevinin‐2‐RN1) and GAFGNFLKGVAKKAGLKILSIAQCKLFGTC (brevinin‐2‐RN2), respectively, by Edman degradation. Different from brevinin‐2, which is composed of 33 amino acid residues (aa), both brevinin‐2‐RN1 and ‐RN2 contain 30 aa. Five cDNA sequences (Genbank accession numbers, EU136465‐9) encoding precursors of brevinin‐2‐RN1 and ‐RN2 were screened from the skin cDNA library of R. nigrovittata. These precursors are composed of 72 aa including a predicted signal peptide, an acidic spacer peptide, and a mature brevinin‐2‐RN. Both brevinin‐2‐RN1 and ‐RN2 showed strong antimicrobial activities against gram‐positive and gram‐negative bacteria and fungi. The current work identified and characterized two novel antimicrobial peptides with unique primary structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses

Innate immune mechanisms of defense are especially important to ectothermic vertebrates in which adaptive immune responses may be slow to develop. One innate defense in amphibian skin is the release of abundant quantities of antimicrobial peptides. Chytridiomycosis is an emerging infectious disease of amphibians caused by the skin fungus, Batrachochytrium dendrobatidis . Susceptibility to chytridiomycosis varies among species, and mechanisms of disease resistance are not well understood. Previously, we have shown that Australian and Panamanian amphibian species that possess skin peptides that effectively inhibit the growth of B. dendrobatidis in vitro tend to survive better in the wild or are predicted to survive the first encounter with this lethal pathogen. For most species, it has been difficult to experimentally infect individuals with B. dendrobatidis and directly evaluate both survival and antimicrobial peptide defenses. Here, we demonstrate differences in susceptibility to chytridiomycosis among four Australian species ( Litoria caerulea, Litoria chloris, Mixophyes fasciolatus and Limnodynastes tasmaniensis ) after experimental infection with B. dendrobatidis , and show that the survival rate increases with the in vitro effectiveness of the skin peptides. We also observed that circulating granulocyte, but not lymphocyte, counts differed between infected and uninfected Lit. chloris . This suggests that innate granulocyte defenses may be activated by pathogen exposure. Taken together, our data suggest that multiple innate defense mechanisms are involved in resistance to chytridiomycosis, and the efficacy of these defenses varies by amphibian species.     

Metamorphic changes in localization of sugars in skin of the leopard frog,Rana pipiens

A lectin histochemical study was carried out to determine the distribution of specific sugars in glycoconjugates within an important osmoregulatory organ, amphibian skin. Paraffin sections were made of Rana pipiens skin from dorsal and ventral regions of aquatic larvae in representative developmental stages as well as from several body regions of semiaquatic adult frogs. Sections were incubated with horseradish peroxidase (HRP)‐conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates. Such sites were visualized by DAB‐H₂O₂. The following HRP‐lectins were used: UEA‐1 for α‐L ‐fucose, SBA for N‐acetyl‐D ‐galactosamine, WGA for N‐acetyl‐β‐D ‐glucosamine, PNA for β‐galactose, and Con A for α‐mannose. We found that lectin binding patterns in larvae change during metamorphic climax as the skin undergoes extensive histological remodeling; this results in adult skin with staining patterns that are specific for each lectin and are similar in all body regions. Such findings in R. pipiens provide additional insight into the localization of molecules involved in osmoregulation in amphibian skin. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Nine polymorphic microsatellite loci for the northern leopard frog (Rana pipiens)

We describe the cloning and characterization of nine microsatellite loci from the northern leopard frog. Seven loci consist of tetranucleotide repeats, one locus consists of a dinucleotide repeat and one locus consists of a GT repeat juxtaposed with a GATA repeat. In a sample of 36 frogs from a natural population, polymorphism at these loci ranged from two to 13 alleles per locus with expected heterozygosities ranging from 0.5 to 0.91. These loci will be useful to researchers since this species is used for a broad range of studies.     

Large-scale seasonal variation in the prevalence and severity of chytridiomycosis

The chytrid fungus Batrachochytrium dendrobatidis has been implicated as the causative agent of mass mortalities, population declines and the extinctions of amphibian species worldwide. Although several studies have shown that the prevalence of chytridiomycosis (the disease caused by the fungus) increases in cooler months, the magnitude and timing of these seasonal fluctuations have yet to be accurately quantified. We conducted disease sampling in a single population of stony creek frogs Litoria wilcoxii on 13 occasions over a 21-month period and used quantitative real-time polymerase chain reaction to detect and quantify the number of B. dendrobatidis zoospores present on samples. Disease prevalence varied significantly across sampling sessions, peaking at 58.3% (in early spring) and dropping to as low as 0% on two occasions (late summer and early autumn). There was a significant negative relationship between disease prevalence and mean air temperature in the 30 days prior to sampling. These large-scale seasonal fluctuations in chytridiomycosis levels will strongly influence conservation programs and amphibian disease research.     

Altitudinal distribution of chytrid (Batrachochytrium dendrobatidis) infection in subtropical Australian frogs

The disappearance of amphibian populations from seemingly pristine upland areas worldwide has become a major focus of conservation efforts in the last two decades, and a parasitic chytrid fungus, Batrachochytrium dendrobatidis, is thought to be the causative agent of the population declines. We examined the altitudinal distribution of chytrid infections in three stream‐dwelling frog species (Litoria wilcoxii, L. pearsoniana and L. chloris) in southeast Queensland, Australia, and hypothesized that if B. dendrobatidis were responsible for the disappearance of high‐altitude frog populations, infection prevalence and intensity would be greatest at higher altitudes. Overall, 37.7% of the 798 adult frogs we sampled were infected with B. dendrobatidis, and infections were found in all the populations we examined. Contrary to our initial hypothesis, we found no consistent evidence that high‐altitude frogs were more likely to be infected than were lowland frogs. Further, the intensity of fungal infections (number of zoospores) on high‐altitude frogs did not differ significantly from that of lowland frogs. Batrachochytrium dendrobatidis appears to be capable of infecting frogs at all altitudes in the subtropics, suggesting that all populations are at risk of decline when conditions favour disease outbreaks. We did find evidence, however, that chytrid infections persist longer into summer in upland as compared with lowland areas, suggesting that montane amphibian populations remain susceptible to disease outbreaks for longer periods than do lowland populations. Further, we found that at high altitudes, temperatures optimal for chytrid growth and reproduction coincide with frog metamorphosis, the life‐stage at which frogs are most susceptible to chytrid infections. While these altitudinal differences may account for the differential population‐level responses to the presence of B. dendrobatidis, the reason why many of southeast Queensland's montane frog populations declined precipitously while lowland populations remained stable has yet to be resolved.     

Effective population sizes and temporal stability of genetic structure in Rana pipiens, the northern leopard frog

Although studies of population genetic structure are very common, whether genetic structure is stable over time has been assessed for very few taxa. The question of stability over time is particularly interesting for frogs because it is not clear to what extent frogs exist in dynamic metapopulations with frequent extinction and recolonization, or in stable patches at equilibrium between drift and gene flow. In this study we collected tissue samples from the same five populations of leopard frogs, Rana pipiens, over a 22-30 year time interval (11-15 generations). Genetic structure among the populations was very stable, suggesting that these populations were not undergoing frequent extinction and colonization. We also estimated the effective size of each population from the change in allele frequencies over time. There exist few estimates of effective size for frog populations, but the data available suggest that ranid frogs may have much larger ratios of effective size (Ne) to census size (Nc) than toads (bufonidae). Our results indicate that R. pipiens populations have effective sizes on the order of hundreds to at most a few thousand frogs, and Ne/Nc ratios in the range of 0.1-1.0. These estimates of Ne/Nc are consistent with those estimated for other Rana species. Finally, we compared the results of three temporal methods for estimating Ne. Moment and pseudolikelihood methods that assume a closed population gave the most similar point estimates, although the moment estimates were consistently two to four times larger. Wang and Whitlock's new method that jointly estimates Ne and the rate of immigration into a population (m) gave much smaller estimates of Ne and implausibly large estimates of m. This method requires knowing allele frequencies in the source of immigrants, but was thought to be insensitive to inexact estimates. In our case the method may have failed because we did not know the true source of immigrants for each population. The method may be more sensitive to choice of source frequencies than was previously appreciated, and so should be used with caution if the most likely source of immigrants cannot be identified clearly.     

Identification and characterization of two novel antimicrobial peptides,temporin‐Ra and temporin‐Rb,from skin secretions of the marsh frog (Rana ridibunda)

In this study, two novel antimicrobial peptides from the skin secretions of the marsh frog, Rana ridibunda, named temporin‐Ra and temporin‐Rb, were identified and purified using RP‐HPLC. Temporin‐Ra and temporin‐Rb are composed of 14 and 12 amino acids, respectively. Our results show that these peptides have inhibitory effects on both gram‐negative and gram‐positive bacteria, especially antibiotic resistant strains prevalent in hospitals, such as Staphylococcus aureus and Streptococcus agalactiae. The sequences and molecular weights of these peptides were determined using tandem MS. The molecular masses were found to be 1242.5 Da for temporin‐Rb and 1585.1 Da for temporin‐Ra. Human red blood cells tolerated well exposure to temporin‐Ra and temporin‐Rb, which, at a concentration of 60 µg/ml, induced 1.3% and 1.1% hemolysis, respectively. MIC values of these peptides are suitable for potent antimicrobial peptides. The low hemolytic effect and wide‐spectrum antimicrobial activity suggest a possible therapeutic application of these novel peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

The physiology of hibernation in Canadian leopard frogs (Rana pipiens) and bullfrogs (Rana catesbeiana)

Canadian northern leopard frogs (Rana pipiens) and bullfrogs (Rana catesbeiana) were acclimated to 3 degrees C and submerged in anoxic (0-5 mmHg) and normoxic (Po(2) approximately 158 mmHg) water. Periodic measurements of blood Po(2), Pco(2), and pH were made on samples taken anaerobically from subsets of each species. Blood plasma was analyzed for [Na(+)], [K(+)], [Cl(-)], [lactate], [glucose], total calcium, total magnesium, and osmolality. Blood hematocrit was determined, and plasma bicarbonate concentration was calculated. Both species died within 4 d of anoxic submergence. Anoxia intolerance would rule out hibernation in mud, which is anoxic. Both species survived long periods of normoxic submergence (R. pipiens, 125 d; R. catesbeiana, 150 d) with minimal changes in acid-base and ionic status. We conclude that ranid frogs require a hibernaculum where the water has a high enough Po(2) to drive cutaneous diffusion, allowing the frogs to extract enough O(2) to maintain aerobic metabolism, but that an ability to tolerate anoxia for several days may still be ecologically meaningful.     

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio

Eight peptides with differential growth–inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH₂) and temporin-1Gd (FILPLIASFLSKFL.NH₂) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC₅₀=2.4±0.1 μM for temporin-1Gb and 2.3±0.2 μM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.     

中国林蛙皮肤抗菌肽基因的cDNA克隆及抗菌、抗癌和溶血活性的测定

采用RT-PCR和3￠RACE方法, 从中国林蛙皮肤总RNA中克隆出了6条编码不同抗菌肽前体的cDNA序列, 分别命名为: preprotemporin-1CEa、preprotemporin-1CEb、preprotemporin-1CEc、preprobrevinin-1CEa、preprobrevinin-1CEb和preprochensinin-1。cDNA碱基序列全长为289~315 bp, 编码59~65个氨基酸。6个抗菌肽前体由3部分结构域组成: 22个氨基酸残基组成的信号肽、多个酸性氨基酸残基组成的中间序列、高度变异的成熟肽。preprotemporin-1CEa、preprotemporin-1CEb和preprotemporin-1CEc属于temporin-1家族抗菌肽前体, 具有肽链短, C-端酰胺化的特点; preprobrevinin-1CEb和preprobrevinin-1CEa属于brevinin-1家族抗菌肽前体, 在肽链的C-端含有RANA盒结构, 可在2个半胱氨酸残基间形成二硫键, 组成7肽环; preprochensinin-1在已知多种数据库中没有发现序列同源的多肽, 被鉴定为新抗菌肽。人工合成temporin-1CEa、temporin-1CEb、brevinin-1CEa和chensinin-1四种中国林蛙皮肤抗菌肽, 活性检测结果表明: 它们对金黄葡萄球菌等3种革兰氏阳性细菌的生长具有明显抑制作用, 同时抑制乳腺癌MCF-7细胞和宫颈癌HeLa细胞生长。     

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.     

Comparison of isometric contractile properties in hindlimb extensor muscles of the frogs Rana pipiens and Bufo marinus: functional correlations with differences in hopping performance

The leopard frog (Rana pipiens) is an excellent jumper that can reach high take-off velocities and accelerations. It is diurnal, using long, explosive jumps to capture prey and escape predators. The marine toad (Bufo marinus) is a cryptic, nocturnal toad, typically using short, slow hops, or sometimes walking, to patrol its feeding area. Typical of frogs with these different locomotor styles, Rana has relatively long hindlimbs and large (by mass) hindlimb extensor muscles compared to Bufo. We studied the isometric contractile properties of their extensor muscles and found differences that correlate with their different hopping performances. At the hip (semimembranosus, SM), knee (peroneus, Per) and ankle (plantaris longus, PL), we found that Rana's muscles tended to produce greater maximum isometric force relative to body mass, although the difference was significant only for PL. This suggests that differences in force capability at the ankle may be more important than at other joints to produce divergent hopping performances. Maximum isometric force scaled with body mass so that the smaller Rana has relatively larger muscles and force differences between species may reflect size differences only. In addition, Rana's muscles exhibited greater passive resistance to elongation, implying more elastic tissue is present, which may amplify force at take-off due to elastic recoil. Rana's muscles also achieved a higher percentage of maximum force at lower stimulus inputs (frequencies and durations) than in Bufo, perhaps amplifying the differences in force available for limb extension during natural stimulation. Twitch contraction and relaxation times tended to be faster in Rana, although variation was great, so that differences were significant only for Per. Fatigability also tended to be greater in Rana muscles, although, again, values reached significance in only one muscle (PL). Thus, in addition to biomechanical effects, differences in hopping performance may also be determined by diverse physiological properties of the muscles.     

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.     

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.     

Isolation and characterization of microsatellite markers in the lowland leopard frog (Rana yavapaiensis) and the relict leopard frog (R. onca), two declining frogs of the North American desert southwest

We characterized 15 microsatellite loci for the lowland leopard frog (Rana yavapaiensis) and the relict leopard frog (R. onca) for future studies of population genetic structure and relatedness. Analysis of 20 individuals from single populations of each species showed that all markers were polymorphic in at least one species. Observed and expected heterozygosities ranged from 0 to 0.94 and from 0.11 to 0.85, respectively, and there were three to 11 alleles per locus. No loci were in linkage disequilibrium, but six loci deviated significantly from Hardy-Weinberg equilibrium, and the presence of a null allele was detected in two of these loci.     

Opposite effects of cooling on twitch contractions of skeletal muscle isolated from tropical toads (Leptodactylidae) and northern frogs (Ranidae)

Cooling increases the twitch force of frog skeletal muscle (Rana temporaria; Rana pipiens), but decreases the twitch force of tropical toad muscle (Leptodactylus insularis). Action potentials and intramembranous charge movement in frog and toad fibers were slowed identically by cooling. Cooling increased the integral of twitch Ca²⁺ detected by aequorin in frog fibers (1.4-fold), while also decreasing the peak and slowing the rate of decay. Conversely, cooling decreased the integral (0.6-fold) and the peak of twitch Ca²⁺ in toad fibers, without affecting the rate of decay. The difference in entire Ca²⁺ transients may account for cold-induced twitch potentiation in frogs and twitch paralysis in toads. In sustained contractions of toad fibers, cooling markedly decreased maximum force caused by: (i) tetanic stimulation, (ii) two-microelectrode voltage clamp steps, (iii) high [K⁺], or (iv) caffeine. Maximum force in sustained contractions was decreased moderately by cooling frog fibers. Rapid rewarming and simultaneous removal of high [K⁺] or caffeine during a sustained contraction, caused toad muscle force to rise towards the value corresponding to the warm temperature. This did not occur after removing high [K⁺] or caffeine from toad fibers kept in the cold. Transmission electron micrographs showed no relevant structural differences. Parvalbumins are thought to promote relaxation of frog muscle in the cold. The unique parvalbumin isoforms in toad muscle apparently lack this property. Accepted: 27 August 1998     

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and North American Rana frogs share the same families of skin antimicrobial peptides

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and the North American pickerel frog (Rana palustris) occupy different ecological niches on two different continents with no overlap in geographical distribution. R. palustris skin secretions contain a formidable array of antimicrobial peptides including homologs of brevinin-1, esculentin-1, esculentin-2, ranatuerin-2, a temporin and a family of peptides considered of unique structural attributes when isolated, palustrins 1-3. Here we describe the structures of mature peptides and precursors of eight putative antimicrobial peptides from the skin secretion of the Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis). Each peptide represents a structural homolog of respective peptide families isolated from R. palustris, including two peptides identical in primary structure to palustrin 1c and palustrin 3b. Additionally, two peptides were found to be structural homologs of ranatuerin 2B and ranatuerin 2P from the closely-related North American species, Rana berlandieri (the Rio Grande leopard frog) and Rana pipiens (the Northern leopard frog), respectively. Both palustrins and ranatuerins have hitherto been considered unique to North American ranid frogs. The use of primary structures of amphibian skin antimicrobial peptides is thus questionable as a taxonomic device or alternatively, the micro-evolution and/or ancestry of ranid frogs is more highly complex than previously thought.     

Heritability of Batrachochytrium dendrobatidis burden and its genetic correlation with development time in a population of Common toad (Bufo spinosus)

Despite the important threat that emerging pathogens pose for the conservation of biodiversity as well as human health, very little is known about the adaptive potential of host species to withstand infections. We studied the quantitative genetic architecture responsible for the burden of the fungal pathogen Batrachochytrium dendrobatidis in a population of common toads in conjunction with other life‐history traits (i.e., body size and development rate) that may be affected by common selective pressures. We found a significant heritable component that is associated with fungal burden, which may allow for local adaptation to this pathogen to proceed. In addition, the high genetic correlation found between fungal burden and development time suggests that both traits have to be taken into account in order to assess the adaptive response of host populations to this emerging pathogen.