Genetic diversity in mitochondrial 12S rDNA of western Palearctic water frogs (Anura, Ranidae) and implications for their systematics

The nucleotide sequence of a part of the mitochondrial 12S rRNA gene of eight western Palearctic water frog species was analysed. The results are consistent with the species status of Rana bedriagae, Rana bergeri, Rana epeirotica, Rana lessonae, Rana perezi, Rana ridibunda, Rana saharica and Rana shqiperica . The obtained DNA data suggest that lake frogs from Greece and Yugoslavia on the one hand and lake frogs from Georgia, Uzbekistan and Turkmenistan on the other hand represent two distinct species. However, it is not yet clear whether lake frogs from Georgia, Uzbekistan and Turkmenistan belong to R. ridibunda or represent a new species. The very high similarity between the analysed 12S rDNA segments of German R. ridibunda and R. lessonae confirm the finding that mtDNA of R. lessonae was transmitted into the mitochondrial gene pool of R. ridibunda probably as a result of backcrosses with the hybridogenetic hybrid R. kl. esculenta . The results of parsimony analyses speak in favour of very close phylogenetic relations between R. perezi and R. saharica ; with a high probability these species represent an adelphotaxon. Furthermore, the clades ( R. lessonae + R. shqiperica + R. bergeri ) and ( R. ridibunda + R. bedriagae ) are considered to be sister groups. According to the mt 12S rDNA data R. epeirotica seems to be more closely related to the supraspecific taxon ( R. ridibunda + R. bedriagae ) than to ( R. lessonae + R. shqiperica + R. bergeri ). Thus, it can be excluded that R. shqiperica and R. epeirotica represent sister species.     

补偿生长对中国林蛙肝脏生化组成的影响

在室内环境条件下研究了不同饥饿程度和再投喂后中国林蛙肝脏中蛋白质、糖原和脂肪含量的变化,并对中国林蛙在补偿生长过程中能量来源的变化进行了分析.结果 表明,饥饿状态下,中国林蛙的能量消耗主要来源于肝糖原,其次是蛋白质,即饥饿时中国林蛙首先动用糖原和蛋白质提供能量.     

Character convergence in advertisement call and mate choice in two genetically distinct water frog hybridogenetic lineages (Rana kl esculenta, Rana kl grafi)

Patterns of advertisement call were investigated in two genetically distinct water frog lineages ( Rana kl esculenta, Rana. kl grafi ), which were identified by starch gel electrophoresis – the aim being to determinate the role of vocalization in the hybridogenetic process. Both hybrids displayed major modifications from the basic structure of the Rana ridibunda call. In Rana kl grafi , the call structure tended to correspond to that of Rana perezi in most of the studied parameters (frequency, duration, number of pulses) whereas the call of Rana kl esculenta tended to resemble that of Rana lessonae . The ascendant hierarchical classification clearly revealed such a convergence toward parental species and accounted for a divergence between hybrids. Changes in call patterns might result from both the expression of the non- ridibunda genome and the sexual selective pressure through female mate choice. Only few non- ridibunda females exhibited a preference for hybrid calls which, however, allowed some hybridogenetic males to obtain successful mates. Thus, hybridogenesis induced character convergence in courtship signal with the non- ridibunda species in hybrid zones. In any case, these changes in the courtship signal favoured the particular hybridogenetic process, which constitutes a quasi-parasitism of the non- ridibunda genome.     

Seasonal variations in frog liver glycogen metabolism (author's transl)

Glycogen metabolism in frog (Rana ridibunda) liver is subject to seasonal variations. Hepatic glycogen and glycogen synthase levels are highest in the fall and winter months and lowest in the summer months, whereas glycogen phosphorylase activity is highest in spring and summer and lowest in fall and winter months. Blood glucose levels show a clear increase during the months of March, June-July and November over the mean level for the rest of year (19.0 +/- 5.5 mg glucose/100 ml serum). Results indicate that the animal accumulated glycogen in the fall to be consumed during the winter. Glycogen levels are in direct proportion to glycogen synthase activity levels (I-form and total activity) and in inverse proportion to glycogen phosphorylase (phosphorylated form) activity levels, which would suggest that these enzymes exercise a direct control over glycogen levels.     

Introducing water frogs--is there a risk for indigenous species in France?

The ecological success of introduced species in their new environments is difficult to predict. Recently, the water frog species Rana ridibunda has raised interest, as different genetic lineages were introduced to various European countries. The aim of the present study was to analyze the potential invasiveness of R. ridibunda and assess the risk of replacement for indigenous water frog species. The investigation of over 700 water frogs from 22 locations in southern France and four locations in Spain shows that the competition with indigenous species is mainly limited to a particular habitat type, characterized by high-oxygen and low-salinity freshwater. The competitive strength of R. ridibunda may be related to a higher growth rate and longevity as compared to the indigenous species R. grafi and R. perezi. Our data suggest that R. ridibunda is a risk to the diversity of indigenous water frog assemblages in France. Future monitoring needs to clarify the distribution of R. ridibunda, its ecological niche, and the risk status for indigenous water frog species.     

Studies on the cytophysiology of the fat body of the American silkmoth

Summary A developmental study at the electron microscopic level was conducted of the fat body cells of Hyalophora cecropia (L.). During the last larval instar the fat body increases in volume and the cells exhibit a well developed rough endoplasmic reticulum and protein bodies of diverse sizes. In the pupal fat body, the protein bodies appear to be enclosed by a double membrane and contain glycogen granules, ribosomes and mitochondrion-like structures. In addition, there are large lipid globules, cytolysomes and rough endoplasmic reticulum. The ultrastructure of the protein bodies suggests the development of large bodies by fusion of smaller protein bodies. Changes in fat body cell ultrastructure were followed during adult development and cytological evidence was obtained for the depletion of protein, glycogen and lipid in the female during this period. The female adult fat body cell contains free ribosomes, protein bodies, many mitochondria, a few lipid globules and glycogen granules. The male moth fat body cells have many mitochondria, a few glycogen granules, essentially no protein bodies, but an abundance of large lipid globules.Studies on the influence of egg maturation on the morphology of the fat body of Hyalophora gloveri (L.) revealed that ovariectomy of pupae yielded adults having more fat body than normal females, and that the fat body cells of the ovariectomized animals contained more glycogen, lipid and protein. Male pupae receiving ovarian implants developed into adults containing eggs and possessed more fat body than normal females but less than normal males. Very few glycogen granules were found in the fat body cells of normal males or males with implanted ovaries.Supported by grant AM-02818 from the National Institutes of Health.We thank Dr. James Oschman for his helpful suggestions and constructive criticisms.     

Lipid levels and lipid peroxidation in frog tissues during hyperthermia and hibernation

The content of cholesterol, phospholipids, free fatty acids in the blood, ketone bodies and lipid peroxidation in the liver, brain, myocardium, skeletal muscles of Rana ridibunda were studied. Changes in the lipid content in blood during artificial hypothermia differ from those during hibernation and arousal. The utilization of reserve fats during hibernation is limited, but significantly rises during arousal. The ability to spontaneous lipid peroxidation under conditions of homogenate incubation at 37 degrees C does not increase in the studied tissues during hibernation.     

The function of fat bodies in relation to the hypothalamo-hypophyseal-gonadal axis in the frog,Rana esculenta

In this study the authors have tried to furnish experimental support for the importance of fat bodies in the normal functioning of the hypothalamo-hypophyseal-gonadal system of the male frog, Rana esculenta. These experiments have shown a hypothalamo-hypophyseal control of the mobilization of fat body contents, directly involved in the control of testicular activity. Furthermore it is proposed that the fat body contents are released into the testis through direct vascular contacts between the two organs. We suggest that the A1 cells (lactotrophs) and/or B2 cells (FSH-gonadotrops) of the pars distalis gonadotropins are incapable of stimulating the testis in the absence of fat bodies. In the light of these results a scheme has been put forward showing the position of fat bodies in the hypothalamo-hypophyseal-gonadal axis of the frog.     

Genome elimination in diploid and triploid Rana esculenta males: cytological evidence from DNA flow cytometry

Cytological aspects of hemiclonal (meroclonal) inheritance in diploid and triploid males of the hybridogenetic frog Rana esculenta (Rana ridibunda x Rana lessonae) have been studied by DNA flow cytometry. The fact that the R. ridibunda genome contains 16% more DNA than the R. lessonae genome provides the ability to discern cells containing genomes of any species from the water-frog complex under study. Data are presented showing that elimination of the R. ridibunda genome occurs in hybridogenetic males from certain populations. In triploid males, the cytogenetic mechanism of hemiclonal inheritance is simpler than in diploids: after the elimination of a genome (always the genome in the minority in the triploid set; "homogenizing elimination"), no compensatory duplication of the remaining genetic material is necessary, as it is in diploids. The process of elimination can be visualized in triploid males by using DNA flow cytometry to identify cells in the special phase of the spermatogonial cell cycle that we termed the E phase.     

Effects of adenosine perfusion on the metabolism and contractile activity of Rana ridibunda heart

The effects of adenosine were examined on the isolated perfused heart of the frog Rana ridibunda. Adenosine produced negative chronotropic and inotropic effects on frog ventricle in a concentration-dependent manner. The effects of adenosine on cardiac metabolism were also investigated by measuring the tissue content of adenine nucleotides, lactate, pyruvate, adenosine and inorganic phosphate, during adenosine perfusion. Adenosine had no effect on the tissue content of metabolites. No net synthesis of adenine nucleotides was observed during perfusion with increasing concentrations of adenosine. Lactate output from the heart decreased significantly with adenosine perfusion. Correlation of adenosine effects on cardiac muscle with the effects of hypoxia are discussed.     

Alpha-melanotropin in the frog brain: regional distribution, quantification and subcellular distribution

The distribution of alpha-MSH containing neurons was studied by immunofluorescence in the brain of the frog Rana ridibunda. Most immunoreactive cell bodies were found in the ventral hypothalamic area. A rich network of fluorescent fibers was observed in the ventral infundibular region, coursing towards the preoptic area and the ventral telencephalon. Some fibers, directed backwards, project into median eminence. By means of a specific radioimmunoassay, the concentrations of alpha-MSH immunoreactive material has been determined in 10 different regions of the brain. The highest concentrations were observed in the infundibular and the preoptic regions. Using the immunogold technique, electron microscopy showed that immunostaining was restricted to 70-100 nm dense core vesicles in positive cell bodies and fibers. These results suggest that, in addition to well known hormonal (melanotropic) activity, alpha-MSH could play the role of a neurotransmitter in the frog brain.     

Freezing tolerance of the European water frogs: the good, the bad, and the ugly

Survival and some physiological responses to freezing were investigated in three European water frogs (Rana lessonae, Rana ridibunda, and their hybridogen Rana esculenta). The three species exhibited different survival times during freezing (from 10 h for R. lessonae to 20 h for R. ridibunda). The time courses of percent water frozen were similar; however, because of the huge differences in body mass among species (from 10 g for Rana lessonae to nearly 100 g for Rana ridibunda), the ice mass accumulation rate varied markedly (from 0.75 +/- 0.12 to 1.43 +/- 0.11 g ice/h, respectively) and was lowest in the terrestrial hibernator Rana lessonae. The hybrid Rana esculenta exhibited an intermediate response between the two parental species; furthermore, within-species correlation existed between body mass and ice mass accumulation rates, suggesting the occurrence of subpopulations in this species (0.84 +/- 0.08 g ice/h for small R. esculenta and 1.78 +/- 0.09 g ice/h for large ones). Biochemical analyses showed accumulation of blood glucose and lactate, liver glucose (originating from glycogen), and liver alanine in Rana lessonae and Rana esculenta but not in Rana ridibunda in response to freezing. The variation of freeze tolerance between these three closely related species could bring understanding to the physiological processes involved in the evolution of freeze tolerance in vertebrates.     

Distribution of atrial natriuretic factor-like immunoreactivity in the brain of the frog Rana ridibunda

The localization of atrial natriuretic factor (ANF)-like immunoreactivity in the central nervous system of the frog Rana ridibunda was examined by the indirect immunofluorescence technique, using an antiserum against synthetic ANF (Arg101-Tyr126). Immunoreactive cell bodies were principally found in the dorsal and medial pallium, the medial septal nucleus, the ventrolateral and anteroventral areas of the thalamus, the lateral forebrain bundle, the posterolateral thalamic nuclei, the preoptic nucleus, the dorsal infundibular nucleus, and the anteroventral tegmentum nucleus of the mesencephalon. Numerous cell bodies and a very dense fiber bundle were visualized in the interpeduncular nucleus. All the areas mentioned above contained a high density of immunoreactive fibers. In addition, the amygdala, the infundibular nucleus, the median eminence, and most of the areas of the mesencephalon contained a moderate number of ANF-positive nerve processes. In the frog pituitary, fibers and nerve terminals were found in the peripheral zone of the neural lobe. The intermediate and anterior lobes of the frog pituitary were totally devoid of ANF immunoreactivity. These results indicate that ANF-like material is widely distributed in the frog brain and that ANF may be involved in various brain functions including neuroendocrine regulations.     

The use of a serological technique to examine host selection in a natural population of the medicinal leech, Hirudo medicinalis

SUMMARY. 1. Serological examinations were made of the bloodmeals of medicinal leeches, Hirudo medicinalis L., collected from two sites at Dungeness, Kent, in order to determine their host range.
2. A modified miniature Ouchterlony agar diffusion method was used with antisera raised in rabbits against fish, bird, frog and mammal blood sera.
3. The results suggest that the marsh frog. Rana ridibunda Pall., was the most important host with smaller contributions from birds and fishes. Mammals appeared to be very rare hosts. These results reflect the availability of hosts as determined by field studies. Many leeches showed evidence of blood meals from more than one host.
4. Field observations indicated the importance of the smooth newt, Triturus vulgaris L., as a host for the leech, particularly early in the season. Predation on the newt appeared invariably to be fatal.     

Comparative characteristics of the vitreous body proteins in vertebrates

Using disc-electrophoresis in polyacrylamide gel and immunochemical methods, studies have been made on proteins from the vitreous body of mammals (albino mouse, rat, guinea pig, pig, dog, cat), birds (hen), amphibians (the frog Rana ridibunda) and fish (the perch Perca fluviatilis). It was found that vitreous body proteins in man and animals include both the specific proteins and those of the blood serum. During evolution, specific antigens of the vitreous body attained strict species specificity, although some of them preserved the initial properties.     

Hemiclone diversity in the hybridogenetic frog Rana esculenta outside the area of clone formation: the view from protein electrophoresis

European water frog hybrids Rana esculenta reproduce hemiclonally, by hybridogenesis: In the germ line they exclude the genome of the parental species Rana lessonae and produce haploid, unrecombined gametes with a genome of the parental species Rana ridibunda . These hybrids coexist with and depend as sexual parasites on the host parental species R. lessonae (the L-E population system); matings with R. lessonae restore somatic hybridity in each generation of R. esculenta . We investigated 15 L-E system populations in northern Switzerland, which is outside R. ridibunda 's native range. Frequency of hybrids in samples varied from 8% in marsh ponds to 100% in gravel pits and forest ponds. Clonal diversity (variation among R. ridibunda genomes of hybrids), detected by six protein electrophoretic marker loci, revealed a total of eight hemiclones and locally ranged from uniclonal populations in southern parts of the survey region to six coexisting hemiclones in the north. All alleles distinguishing hemiclones occur commonly in the nearest native R. ridibunda populations of east-central Europe; the most probable source of clonal diversity in our samples is multiple clone formation by primary hybridizations in the sympatry area of R. ridibunda and R. lessonae and subsequent dispersal of hemiclonal lineages. A positive correlation between amount of clonal diversity and hybrid frequency, predicted by the Frozen Niche Variation (FNV) model (each hemiclone is characterized by a relatively narrow niche, coexistence is possible through niche partitioning), was not found; this contrasts with hemiclonally reproducing fish hybrids ( Poeciliopsis ). Historical factors, such as availability of different colonizing hemiclones may be strong enough to override the signal from operation of the FNV.     

Oxygen consumption of a terrestrial toad (Bufo viridis) and semi-aquatic frog (Rana ridibunda)

1. Oxygen consumption was measured before and after dehydration at different ambient temperatures (Ta) in the terrestrial toad Bufo viridis and the semi-aquatic frog Rana ridibunda. 2. The metabolic rates at Tas between 14 and 27 degrees C were almost the same for R. ridibunda and B. viridis. 3. The metabolic rate at higher Tas (between 27 and 36 degrees C) was higher in R. ridibunda than in B. viridis. This situation was found before and after dehydration. 4. A similar situation was found with CO2 production, which was higher at high Tas in R. ridibunda compared with B. viridis.     

Biochemical variability and genetic differentiation of marsh frog Rana ridibunda Pall. populations]

Marsh frog populations of Rana ridibunda s. lato from the Ukraine, Caucasus, Turkmenistan and Tadzhikistan have been analyzed genetically by 31 isozyme gene loci. The genetic distance between populations from Tadzhikistan on the one hand, and other regions, on the other hand is D-0.269, that assumes evolutionary divergence at the species level. The presence of fixed allelic substitutions between two populations from Tadzhikistan by five gene loci (Aat-S, Ldh-B, Alb, Es-5) supposes genetic heterogeneity of species in this region. Peculiarities of allozyme variation of this species are discussed.     

Para-aminohippuric acid secretion by the vertebrate kidney

Tubular secretory capacity has been investigated in the kidney of marine fishes Scorpaena porcus, Spicara smaris, frog Rana ridibunda and albino rat by determination of maximal transport of p-aminohippurate--TmPAH, Following values of Tmax expressed in mg/hour per 100 g of body weight were obtained: 0.73 +/- 0.17 for S. porcus, 0.92 +/- 0.09 for S. smaris, 0.93 +/- 0.10 for R. ridibunda and 15.6 +/- 1.6 for rats.