Tachykinin immunoreactivity in the European common frog, Rana temporaria: localization, quantification and chromatographic characterization.

1. Tachykinin immunoreactivity has been localized, quantified and chromatographically-characterized in the brain, stomach, intestine and skin of Rana temporaria. 2. Antisera to mammalian substance P (SP) and neurokinin A (NKA) immunostained nerve fibres in all tissues except skin, and a population of mucosal endocrine cells in the intestinal epithelium. 3. Radioimmunoassay of tissue extracts identified SP immunoreactivity in all tissues but NKA immunoreactivity was restricted to the brain. 4. Chromatographic analysis of both frog tachykinins revealed that they possessed different physico-chemical properties than their mammalian counterparts.     

Genome size variation in the common frog Rana temporaria

Genome size variation in the common frog (Rana temporaria) was investigated with flow cytometry in three latitudinally separated populations in Sweden to see whether it could provide a useful tool for sex-identification in this species. Depending on the sex and population, per cell DNA content (2C value) varied from 8.823 to 11.266 pg with a mean (+/- SE) 2C value of 9.961+/-0.083 pg. Analysis of variance revealed significant differences in genome size among populations and between sexes. Females had ca 3% larger genomes (x=10.133+/-0.068 pg) than males (x=9.832+/-0.068 pg) in all of the populations (sex x population interaction: P>0.10). Individuals from the southern-most population had significantly (x=9.330+/-0.081 pg) smaller genomes than those from the more northern populations (x=10.032+/-0.085 and x=10.584+/-0.085 pg, respectively). These results are in line with the interpretation that males in the common frog are the heterogametic sex, and that there exists large (up to 12%) geographic variation in genome size in this species. However, the sex differences in the genome size are too small to be useful in individual sex identification.     

Pool desiccation and developmental thresholds in the common frog, Rana temporaria

The developmental threshold is the minimum size or condition that a developing organism must have reached in order for a life-history transition to occur. Although developmental thresholds have been observed for many organisms, inter-population variation among natural populations has not been examined. Since isolated populations can be subjected to strong divergent selection, population divergence in developmental thresholds can be predicted if environmental conditions favour fast or slow developmental time in different populations. Amphibian metamorphosis is a well-studied life-history transition, and using a common garden approach we compared the development time and the developmental threshold of metamorphosis in four island populations of the common frog Rana temporaria: two populations originating from islands with only temporary breeding pools and two from islands with permanent pools. As predicted, tadpoles from time-constrained temporary pools had a genetically shorter development time than those from permanent pools. Furthermore, the variation in development time among females from temporary pools was low, consistent with the action of selection on rapid development in this environment. However, there were no clear differences in the developmental thresholds between the populations, indicating that the main response to life in a temporary pool is to shorten the development time.     

GABA immunoreactivity in the main olfactory bulb of the frog Rana temporaria

Immunoreactivity for gamma-aminobutyric acid (GABA) was localized at the light microscopic level in the main olfactory bulb (MOB) of the frog, Rana temporaria. By means of free-floating peroxidase-antiperoxidase immunocytochemical technique, GABA was found in a large number of neurons in the granular cell layer, in a few small somata in the mitral cell layer and in two different types of cell somata in the glomerular layer. Individual GABA-immunopositive cells were found in the olfactory nerve layer. GABA immunostaining was also localized in cell processes and fiber fragments. There were many immunoreactive puncta in all layers of the MOB. GABA-positive punctate structures often outlined immunonegative cells in the mitral cell and glomerular layers. Rounded tightly packed groups of immunoreactive puncta were found only along ventral border of the glomerular layer. The results are discussed in comparison with data obtained on mammalian MOB in terms of MOB functional organization.     

Molecular characterization of major histocompatibility complex class II alleles in the common frog, Rana temporaria

Major histocompatibility complex (MHC) class II genes, which play a major role in the immune system response, are some of the most polymorphic genes in vertebrates. We developed polymerase chain reaction primers for part of the second exon of an expressed MHC class II gene in the common frog, Rana temporaria. We genotyped this locus in five frog populations in southeast England and detected eight alleles in 215 individuals. Five or six alleles were detected in each population with a maximum of two alleles per individual, indicating that only a single locus was amplified. We also inferred the possible existence of a null allele. There were 23 variable nucleotide sites (out of 136) and 13 variable amino acid sites (out of 44), many of which corresponded to amino acids involved in antigen recognition. We detected a significant excess of nonsynonymous substitutions at antigen binding sites, indicating that this gene is under positive selection. The level of variation we found was similar to that in other amphibian MHC class II loci, such as those in Bombina bombina, Xenopus laevis and Ambystoma tigrinum.     

Karyosphere of the oocytes of the common frog, Rana temporaria]

Karyospheres of ca. 200 mcm in diameter were isolated from the common frog oocytes of definitive size. An electron microscope study has revealed in the karyosphere fibrillar nucleoli and micronucleoli, modified synaptinemal complexes sometimes connected with chromatin and fibrillar material containing a great number of, mostly atypical, pore complexes resembling those of nuclear membrane and forming "pseudomembranes". An electrophoresis of the isolated karyosphere has revealed 12 distinct protein bands, of 3 which correspond to the protein triplet characteristic of the nuclear matrix and the rest 9 represent high molecular weight components with the molecular weight from 130 to 200,000 D.     

Refrigerated storage of European common frog Rana temporaria oocytes

Reproduction technologies (RTs) for the storage and use of amphibian gametes have rapidly developed since the recognition of the amphibian conservation crisis in the late 20th Century. Of these RTs, the refrigerated storage of oocytes and sperm can help to achieve reliable pair-matching when unexpected deaths could lead to critical gaps in studbook programs, and also to enable gamete transport between facilities or when sampled from field populations. Viable sperm can be reliably stored in vitro in testes, as suspensions in refrigerators for weeks and in situ in refrigerated carcasses for days. However, oocytes have only been reliably stored in vitro and then only for a few hours. We stored mature oocytes of the European common frog Rana temporaria refrigerated at 4 °C: in situ in the oviduct of carcasses for 1–5 days, in vivo in the oviduct of live frogs for 30 days, and in vitro in plastic boxes for 1–5 days. Oocyte viability was measured as the percentage of fertilisation relative to controls and as the percentage hatch of fertilised oocytes. Rana temporaria oocytes in situ or in vitro retained some viability to hatch for up to 5 days. In contrast, when stored in vivo, oocytes showed little loss of viability to hatch after 10 days and moderate viability up to 30 days.     

Seasonal changes in endocrine organs of the male common frog,Rana temporaria

Summary The annual cycle of the five morphological cell types in the pars distalis of the adenohypophysis of male common frogs,Rana temporaria, was studied in variously stained sections of the pituitary, collected at monthly intervals. All cell types showed a maximal development at the end of hibernation, a strong extrusion of secretory granules in the breeding season, and a more gradual release during later spring and early summer. A minimal development occurred in mid-summer, and a restoration followed in autumn and the hibernation period. During the phase of extrusion production was low, and vice-versa. These seasonal changes correspond with those of the testes and interrenal tissue of the same animals as well as with those of the thyroids and other endocrine tissues, and seems to be an important part of the biphasic annual life cycle of the species.The authors gratefully acknowledge the patient and skilful technical assistance of MissFemmy Brands. They thank Mr.H. van Kooten for making the photographs.     

Determination of growth and maturation in the common frog, Rana temporaria, by skeletochronology

Growth and maturation in a Swiss population of Rana temporaria were studied in 1983 and 1984 by means of skeletochronology. Resting line (growth ring) diameters were used to back-calculate individual body sizes in previous years; these permitted establishment of an average growth curve and determination of individual ages and sizes at first reproduction. Growth was rapid up to maturation, but continued thereafter at a decreased rate. Males were larger than females at age two but females grew faster thereafter, causing sexual dimorphism in adult body sizes. Body size distributions for both years and for frogs recaptured and first captured in 1984 were established. Growth in immatures was positively, but in adults negatively correlated with body size, with considerable variation at all sizes. Individual adult sizes were positively correlated with body sizes at the end of the first year. Average individual age at first reproduction was 2.8 years in males and 3.1 years in females (range in both sexes two to four years). There is no evidence for a two-year-cycle of reproduction.     

MHC zygosity in the frog,Rana temporaria

The major histocompatibility complex (MHC) zygosity of the field-collected frogs, Rana temporaria, was detected by progeny testing. Groups of sibling tadpoles were grafted with intrafamilial tail-tip allografts and the ratio of rapidly rejected allografts to slowly rejected ones was estimated. Twenty-five percent of parental frogs appeared to be MHC homozygotes. Thus, MHC homozygosity in natural frog populations seems to be considerably higher than in wild mouse populations.     

Isolation and characterization of 145 polymorphic microsatellite loci for the common frog (Rana temporaria)

We describe primers and polymerase chain reaction conditions to amplify 145 di‐, tri‐ and tetranucleotide microsatellite loci from the common frog (Rana temporaria), a species commonly used as a model in ecological and evolutionary research. Primers were tested on 46 individuals from two Fennoscandian populations and yielded an average of six to nine alleles per locus (range = 1–30) depending on the population. Average observed heterozygosities in the two populations were 0.16 (range = 0–0.91) and 0.36 (range = 0–1).     

Microsatellite marker data suggest sex-biased dispersal in the common frog Rana temporaria

Despite being important models in ecological, evolutionary and conservation biology research, very little is known about the dispersal in anuran amphibians, and juvenile dispersal in particular. Using microsatellite data, we assessed signatures of sex-biased migration in the common frog (Rana temporaria) in Scandinavia. Significant heterozygosity deficiency (FIS) and lower assignment value (mAIc) among females suggest that dispersal in R. temporaria is female biased. Also variance of assignment (vAIc), estimated separately for the two sexes, was consistent with this inference, although the difference was not statistically significant. Possible proximate and ultimate explanations for female-biased dispersal in amphibians are discussed.     

Effect of photostimulation on the wakefulness-sleep cycle in the common frog Rana temporaria

Effect of daily 30-min photostimulation in the 10 s light: 10 s pause (the total of 5 days) on the time structure of the wakefulness--protosleep cycle (WPC) was studied in the common frog Rana temporaria. Changes were analyzed of EEG wave components in three immobility forms of the type of catalepsy (P-1), catatonia (P-2), and cataplexy (P-3) that form protosleep. The first three photostimulations promoted a gradual increase of the P-1 state to 84.16 +/- 11.6% [the initial value (IV) 22.9 +/- 9.1%] and a decrease of representation of wakefulness to 4.86 +/- 2/1% (IV 13.8 +/- 7.8%), of P-2 to 11.1 +/- 5.3 (IV 53.3 +/- 13.3%), and of P-3 to 2.21 +/- 1.0% (IV 11.1 +/- 5.6%). After 4-5 photostimulations and especially after their complete cessation the percentage of P-1 in the WPC was restored to initial values, whereas the percentage of the frog WPC P-3 considered to be a precursor of the homoiothermal sleep rose to 20 +/- 8.3% after 5 photostimulations and to 38.5 +/- 6.7% the next day. Changes in the frog EEG spectra appeared only after one photostimulation and were characterized by a brief increase of power of alpha-like waves and by inhibition of slow 6-waves. In P-2 the power of the slow delta-waves gradually rose. In P-3 the EEG parameters did not change. In all experimental animals a decrease of the relative thymus and adrenal masses was revealed, which indicates the photostimulation regime used in the work induces stress. The obtained data allow thinking that a certain neurohormonal response to stress has already been formed at the amphibian level and that an important role in this response realization is played by a coordinated interaction of the hypothalamic sleep-regulating system providing protosleep manifestations and of the hypothalamic neurosecretory system triggering the stress-reaction hormonal cascade.     

Tooth replacement in the frog Rana temporaria

The development and replacement of teeth in the frog Rana temporaria is analyzed by dividing the life cycle of the tooth into a number of stages. These stages are identified by the examination of alizarin whole mounts. The dentition in this species is fairly complete and the percentage of functional loci is approximately 74. The teeth in alternate loci are usually at about the same stage in development. The low percentage of non-functional loci is accounted for by the retention of functional teeth over a large fraction of the total life cycle time and the relatively rapid ankylosis of replacement teeth. It is suggested that tooth replacement is essentially a process which involves teeth in alternate loci and that the replacement waves (which connect alternate loci) run parallel to the longitudinal axis of the jaw and are of infinite length. This basic pattern is obscured by many breaks which occur in the replacement waves. The presence of such breaks may be accounted for by variations in the time intervals between the successive stimuli which initiate the Zahnreihen, or simply by the acceleration or deceleration of the development of teeth in one or more loci.     

Using genetic variation to infer associations with climate in the common frog,Rana temporaria

Recent and historical species' associations with climate can be inferred using molecular markers. This knowledge of population and species‐level responses to climatic variables can then be used to predict the potential consequences of ongoing climate change. The aim of this study was to predict responses of Rana temporaria to environmental change in Scotland by inferring historical and contemporary patterns of gene flow in relation to current variation in local thermal conditions. We first inferred colonization patterns within Europe following the last glacial maximum by combining new and previously published mitochondrial DNA sequences. We found that sequences from our Scottish samples were identical to (92%), or clustered with, the common haplotype previously identified from Western Europe. This clade showed very low mitochondrial variation, which did not allow inference of historical colonization routes but did allow interpretation of patterns of current fine‐scale population structure without consideration of confounding historical variation. Second, we assessed fine‐scale microsatellite‐based patterns of genetic variation in relation to current altitudinal temperature gradients. No population structure was found within altitudinal gradients (average F_ST = 0.02), despite a mean annual temperature difference of 4.5 °C between low‐ and high‐altitude sites. Levels of genetic diversity were considerable and did not vary between sites. The panmictic population structure observed, even along temperature gradients, is a potentially positive sign for R. temporaria persistence in Scotland in the face of a changing climate. This study demonstrates that within taxonomic groups, thought to be at high risk from environmental change, levels of vulnerability can vary, even within species.