In vivo generation of 5-lipoxygenase products in frogs and toads

Eicosanoid production by inflammatory cells which resulted from infection of the peritoneal cavity of Rana catesbeiana and Bufo americanus was studied after addition of exogenous arachidonic acid and for metabolites generated in vivo. From exogenous substrate, the cells of Rana catesbeiana produced substantial amounts of 5-hydroxyeicosatetraenoic acid, leukotriene B4, the non-enzymatic isomers of leukotriene B4 and leukotriene C4. From endogenous substrate, 5-hydroxyeicosatetraenoic acid and leukotriene B4 were produced. Cells from Bufo americanus produced leukotriene B4 and 5-hydroxyeicosatetraenoic acid, from both exogenous and endogenous substrate. These observations of in vivo eicosanoid production confirm the participation of 5-lipoxygenase activity in the inflammatory response to infection.     

Effects of pH and aluminium on embryonic and early larval stages of Swedish brown frogs Rana arvalis, R. temporaria and R. dalmatina

The effects of pH and aluminium on embryonic and early larval stages of Swedish brown frogs Rana arvalis, R. temporaria and R. dalmatina were tested in laboratory bioassays. In all three species egg mortality and time needed for embryonic development to hatching increased when pH declined, but no significant effects were found on embryonic development when aluminium level was elevated. In R. arvalis and R. temporaria larval mortality was affected by both pH and aluminium. In both species the frequency of occurrence of larval deformities increased in acid water, and there was a synergistic effect of pH and aluminium. In R. arvalis swimming behaviour was disturbed by high levels of aluminium at pH 5. In all three species the frequency of stressed larvae increased when pH was depressed and aluminium concentration elevated, and there was a synergistic effect when both were combined. The three species differed significantly in egg mortality, time needed for embryonic development, larval mortality, larval deformities and larval stress al low pH and high aluminium levels. R. arvalis showed the highest acid tolerance and R. dalmatina was the most sensitive to low pH.     

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.     

Ammonia as a pheromone in anuran tadpoles

In laboratory experiments, conspecific excretions and ammonia solutions evoked avoidance reactions in tadpoles of three anuran species, Bufo bufo, Rana temporaria, and Rana arvalis. A differential sensitivity of ammonia chemoreception was determined for two anuran species. For Bufo bufo tadpoles, these characteristics at ammonia background concentration of 0.2 mg/l lied in the range 150 % < dI/I < 500 %, and for background of 0.4 mg/l the value lied in the range 400 % < dI/I < 500 %. For Rana temporaria tadpoles, differential threshold against ammonia background concentration of 0.15 mg/l was close to 200 % and against background ammonia concentration of 1.1 mg/l was close to 100 %. These results suggest that such sensitivity of both anurans is sufficient for using ammonia in intra- and interspecies communication.     

Evolutionary relationships of Eastern Palearctic Brown Frogs, genus Rana: paraphyly of the 24-chromosome species group and the significance of chromosome number change

The Eurasian 'brown frogs' are a morphologically conservative assemblage consisting of the European Rana temporaria and a large number of similar species considered to be related. Although the chromosome number of the great majority of Rana species is 26, there are several species of brown frogs (including R. arvalis, R. chensinensis, R. omativentris and R. dybowskii ) which instead have 24 chromosomes. Yet comparative study of isozyme variation at 25 gene loci from these and seven other European, Caucasian, and east Asian species indicates that the 24-chromosome brown frogs are not a monophyletic group. To the exclusion of R. arvalis , the east Asian 24-chromosome species form a distinct clade related to the 26-chromosome species R. temporaria, R. dalmatina, R. japonica , and R. tagai more closely than to R. arvalis. This result is reinforced by the comparative position of the nucleolar organizer region (NOR) in the karyotypes of these frogs, as shown by NOR-banding of metaphase chromosomes. The NOR is on chromosome No. 2 in R. arvalis but on chromosome No. 10 in the other three 24-chromosome species. The systematic positions of the Causasian species A. camerani and R. macrocnemis relative to the other species are unclear except that these frogs are distinct enough to be considered part of an outgroup clade. However, in general, the systematics of the entire brown frog group remains unresolved due to great genetic divergences between species and the evident homoplasy of the brown frog morphotype. The parallel origins of reduced karyotypes among the brown frogs demonstrate that caution should be exercised when judging systematic relationships based upon gross karyology in the absence of more detailed information.     

Influence of introduced fish Percottus glenii (Odontobutidae, Pisces)

Monitoring of 28 waterbodies has been carried out since 1994 in the region of reserve "Lake Glubokoe" (Moscow region, Russia). It was revealed that species diversity as well as abundance of larval amphibians correlate negatively with presence of introduced fish, rotan, Perccottus glenii (Odontobutidae). Newts (Triturus cristatus, T. vulgaris) and frogs (Rana temporaria, R. arvalis, R. lessonae) as a rule are not capable to breed in waterbodies colonised by rotan. In contrast, toads (Bufo bufo) breed successfully in such sites. Larvae of toads are comparatively less edible for rotan and pass their metamorphosis. Persistence of amphibians to predation of rotan decreases in the row: B. bufo (R. temporaria, R. arvalis and R. lessonae), T. vulgaris, T. cristatus. The Crested newt (T. cristatus) is the most endangered species and could extinct in next years.     

Selective predation on tadpoles of three tailless anuran species]

In laboratory experiments an introduced fish Perccottus glenii consumed selectively tadpoles of three numerous anuran amphibian species of Moscow Province. Perccottus glenii actively consumed all seized tadpoles of the Rana arvalis and R. temporaria. These predators consumed significantly lesser number of Bufo bufo tadpoles and frequently rejected them after seizing without considerable damages. Observations showed that P. glenii rejects B. bufo after intraoral testing. Larvae of dragonfly Aeschna cyanea also actively consumed almost all seized R. arvalis tadpoles, while usually rejected B. bufo after attacking and damaging them. Larvae of diving beetle Dytiscus marginalis sucking out captured prey, intensively consumed tadpoles of R. arvalis and B. bufo and did not reject them.     

Dependence of the duration of one mitotic cycle during synchronous cleavage divisions (tau o) on the temperature in 4 Rana species and the limits of the optimal temperatures for their reproduction and early development

The dependence of the duration of one mitotic cycle during synchronous cleavage divisions (tau o) on temperature was studied in Rana temporaria, R. arvalis, R. lessonae, R. ridibunda and the corresponding curves were plotted in both linear and semilogarithmic scale. The curves can be used to characterize the relative duration of development, as well as to estimate the limits of optimal temperatures and to reveal the temperature-temporal regularities of development in these species.     

Quantitative estimation of area parameters (with representatives of genus rana as a case study)

A quantitative method of species "point" area analysis is considered that provides the interpolation of species distribution to the whole territory on the basis of its relationships with climatic and relief variables. It is shown that application of standart statistical interpolation techniques is incorrect. The proposed approach is based on interpolation onto the whole territory the species-specific relations with environmental variables detected in single "points". The basic method for the task proves to be the factor analysis. Within the scope of the study, we have considered the methods for quantitative representation of species relationships with climatic and relief variables. The analysis efficiency is demonstrated by an example of three species of brown frogs: Rana temporaria, R. arvalis u R. amurensis.     

A Comparative Study of the Aminergic Structures in the Hypothalamus and the Hypophysis of Amphibian Larvae,with Special Reference to the Innervation of the Pars Distalis

The distribution and occurrence of the aminergic structures of the hypothalamus and the hypophysis of the investigated species are very similar, except for the pars distalis. In the tadpoles of the anuran species Rana temporaria, R. arvalis, R. dalmatina, R. escutenta, Bufo bufo and B. calamita, the pars distalis is aminergically innervated (the Falck-Hillarp technique), but these nerves disappear at the metamorphic climax. In the larvae of the urodelan species Triturus cristatus and T. vulgaris, the pars distalis is devoid of such nerves. Electron microscopical studies of R. temporaria, B. bufo and T. cristatus confirm these observations. A discussion of the functional significance of the pars distalis nerves and the phylogenetic role of the difference between anuran and urodelan species is given.     

Role of recessive and dominant mutations in adaptation of the genus Rana to modern biosphere

The populations of three anuran amphibian species (Amphibia, Anura) of the genus Rana (R. ridibunda Pall., R. arvalis Nilss. and R. temporaria L.) inhabiting the Yekaterinburg urban agglomeration were examined. The frequencies of two traits, morph striata and iris depigmentation, were estimated in these populations. The former trait, so-called morph striata, is phenotypically expressed as a light dorsomedial stripe. It is controlled by a dominant allele of autosomal diallelic gene striata in some species of Rana genus, exhibiting complete dominance. Striata individuals have a number of physiological features that confer them an advantage under conditions of natural and artificial geochemical anomalies. The second trait, iris depigmentation, is the result of a recessive mutation. The individuals homozygous for this trait have low viability. Thus, the dominant mutations promote rapid adaptation of their carriers. Conversely, the recessive mutations may reduce viability of an individual.     

Microsatellite variation and population structure of the moor frog (Rana arvalis) in Scandinavia

Several recent studies have found amphibian populations to be genetically highly structured over rather short geographical distances, and that the rate of genetically effective dispersal may differ between the sexes. However, apart from the common frog ( Rana temporaria ) little is known about the genetic structuring and sex-biased dispersal in northern European amphibians. We investigated the patterns of genetic diversity and differentiation within and among Scandinavian populations of the moor frog ( Rana arvalis ) using microsatellite markers. The genetic diversity within local R. arvalis populations was not a simple linear negative function of latitude but a convex one: genetic diversity peaked in mid-latitude populations, and declined thereafter dramatically towards the north. The average degree of genetic differentiation among populations ( F _ST = 0.14) was lower than that observed for the common frog ( F _ST = 0.21), though the pattern of isolation by distance was similar for both species. Contrary to common frogs, no evidence for female-biased dispersal was found. The results reinforce the view that amphibian populations are—in general—highly structured over relatively small geographical distances, even in comparatively recently colonized areas.     

Morphological and life history responses to predators in larvae of seven anurans

Amphibian larvae often face two major sources of mortality: pond desiccation and predation. Tadpoles of seven anuran species with different preferences for type of breeding habitat, on a hydroperiod scale, were tested for responses to the presence of predators by raising them experimentally in the presence and absence of a separately caged invertebrate predator that was fed on conspecific tadpoles. The species typically breeding in temporary or semi-permanent ponds ( Rana temporaria , Rana arvalis , Rana dalmatina and Hyla arborea ) – where invertebrate predator populations are predicted to vary considerably spatiotemporally – all showed marked induced increases in tail fin depth in response to predator presence. These species also tended to respond by reduced growth rates. The representative of the most ephemeral habitats, Bufo calamita , did not respond in any of these traits. Its congeneric, Bufo bufo , a toxic inhabitant of permanent ponds and lakes, tended to respond to predator presence by reducing its growth rate, though not by a tail depth increase. I argue that the rather poor swimming performance in Bufo tadpoles may opt for defences other than locomotor ability. The palatable, permanent pond species Pelobates fuscus did not alter either its growth rate or tail morphology. Possible explanations for this result are discussed.     

The introduced fish, rotan (Perccottus glenii), depresses populations of aquatic animals (macroinvertebrates, amphibians, and a fish)

The fish rotan (Perccottus gleniiDybowski) was accidentally introduced into European Russia from the Amur River basin. Rotan is capable of colonising small waterbodies – favourable breeding sites of native amphibians. To reveal its influence on the native aquatic fauna, monitoring of small waterbodies has been carried out since 1994 in the region of Lake Glubokoe Reserve (Moscow Province, Russia). The fish's diet includes a wide range of animal species of all trophic levels. Rotan considerably decreases the species richness of aquatic macroinvertebrates and larval amphibians. As a rule, most amphibian species (Triturus cristatus, T. vulgaris, Rana temporaria, R. arvalis, R. lessonae) and the fish Carassius carassius failed to breed successfully in ponds inhabited by rotan. In contrast, the toad Bufo bufo bred successfully in such sites because its larvae are distasteful to rotan. Rotan–amphibian interactions are discussed.     

Effect of imidazole compounds on the activity of adenosine-3', 5'-monophosphoric acid phosphodiesterase]

The effect of 24 imidazol derivatives on the activity of phosphodiesterase (3', 5'-AMP-phosphohydrolase; KF3.1.4.1) was studied in the experiments with the homogenates of the rat brain and of the Rana temporaria skeletal muscles. Imidazol derivatives could produce both the activating and inhibitory influence on the enzyme. Imidazol and seven of its alkyl-substituted derivatives activated the phosphodiesterase. TTFB (tetrachloro-2-trifluoromethylbenzimidazol) produced the greatest inhibitory effect among the inhibitors on the phosphodiesterase activity.     

Pattern and rate of ovary differentiation with reference to somatic development in anuran amphibians

The rate of somatic development of anuran amphibians is only roughly correlated with the rate of gonad differentiation and varies among species. The somatic stage of a tadpole often does not reflect its age, which seems to be crucial for gonad differentiation rate. We compared the morphology and differentiation of developing ovaries at the light and electron microscopy level, with reference to somatic growth and age of a female. Our observations were performed on 12 species of six families (Rana lessonae, R. ridibunda, R. temporaria, R. arvalis, R. pipiens, R. catesbeiana, Bombina bombina, Hyla arborea, Bufo bufo. B. viridis, Xenopus laevis, Pelobates fuscus) and compared with the results obtained by other authors. This allowed us to describe the unified pattern of anuran female gonad differentiation. Ovary differentiation was divided into 10 stages: I-III, undifferentiated gonad; IV, sexual differentiation; V, first nests of meiocytes; VI, first diplotene oocytes; VII-IX, increasing number of diplotene oocytes and decreasing number of oogonia and nests; X, fully developed ovary composed of diplotene oocytes with rudimental patches of oogonia. We distinguished three types of ovary differentiation rate: basic (most species), retarded (genus Bufo), and accelerated (green frogs of the subgenus Pelophylax genus Rana).     

Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens.

The skins of frogs of the genus Rana synthesize a complex array of antimicrobial peptides that may be grouped into eight families on the basis of structural similarity. A total of 24 peptides with differential growth-inhibitory activity towards the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast Candida albicans were isolated from extracts of the skins of three closely related North American frogs, Rana luteiventris (spotted frog), Rana berlandieri (Rio Grande leopard frog) and Rana pipiens (Northern leopard frog). Structural characterization of the antimicrobial peptides demonstrated that they belonged to four of the known families: the brevinin-1 family, first identified in skin of the Asian frog Rana porosa brevipoda; the esculentin-2 family, first identified in the European frog Rana esculenta; the ranatuerin-2 family, first identified in the North American bullfrog Rana catesbeiana; and the temporin family, first identified in the European frog Rana temporaria. Peptides belonging to the brevinin-2, ranalexin, esculentin-1 and ranatuerin-1 families were not identified in the extracts. Despite the close phylogenetic relationship between the various species of Ranid frogs, the distribution and amino-acid sequences of the antimicrobial peptides produced by each species are highly variable and species-specific, suggesting that they may be valuable in taxonomic classification and molecular phylogenetic analysis.     

Distribution of vasoactive intestinal peptide-like immunoreactivity in the taste organs of teleost fish and frog

Summary Using immunohistochemistry, vasoactive intestinal peptide (VIP) was visualized in taste bud cells of the carp, Cyprinus carpio, and the European catfish, Silurus glanis, by means of light and electron microscopy. Intracellular membrane systems, presumably smooth endoplasmic reticulum, of light (sensory) cells, but not of dark (supporting) cells and basal cells, were densely labelled with antibody. In the frog (four species: Rana temporaria, R. ridibunda, R. arvalis, R. pipiens), taste bud cells did not label. However, the dense basal nerve fibre plexus, some subepithelial ganglionic cells, but no ascending intragemmal fibres, were immunoreactive. In fish, the results support evidence that VIP is involved in the modulation of taste transduction at the level of receptor cells. In the frog, an indirect, possibly vasodilatatory effect on taste perception may be considered.     

NAG infection in Rana temporaria with suppressed normal intestinal microflora]

Frogs of the Rana temporaria species with disturbed biocenosis induced by combined effects of hypothermia (+4 degrees C) and tetracycline were used as an experimental model. The animals were inoculated orally with cultured NAD-vibrios and subjected to clinical, bacteriological, immunomorphological and electron microscopical examinations. The lymph-blood system was shown to be a possible pathway for generalization of the infection. Several causes of long-term persistence of the vibrios in Rana temporaria are discussed.     

Preparation and characterization of two major isotypes of parvalbumins from skeletal muscle of bullfrog (Rana catesbeiana)

Two major isotypes of parvalbumins (PA1 and PA2) have been isolated from the skeletal muscle of bullfrog, Rana catesbeiana. The Mr values were estimated to be 10,100 (PA1) and 11,800 (PA2) by SDS/polyacrylamide gel electrophoresis, and the isoelectric points were determined to be 4.78 (PA1) and 4.97 (PA2) by polyacrylamide gel isoelectric focusing. The amino acid compositions and isoelectric points indicate that PA1 corresponds to Rana esculenta pI 4.50 and Rana temporaria pI 4.75 parvalbumins and PA2 to Rana esculenta pI 4.88 and Rana temporaria pI 4.97 parvalbumins, showing that PA1 is genetically a beta-parvalbumin and PA2 an alpha-parvalbumin. However, in terms of the amino acid compositions, PA1 and PA2 are distinctly different from the corresponding parvalbumins of Rana esculenta or Rana temporaria. The ultraviolet spectra of PA1 and PA2 are consistent with their amino acid compositions. An ultraviolet difference spectrum of the Ca2+-loaded form vs. metal-free form indicates that a Tyr and some Phe residues in PA1 are affected by a conformational change associated with the binding of Ca2+. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the Ca2+-loaded form of PA1 migrated twice as fast as the Mg2+-loaded form. Both PA1 and PA2 show increased mobility in the Ca2+-loaded forms, like troponin C but different from calmodulin.