Interpopulation comparison of variations in gastrulation of <Emphasis Type="Italic">Rana arvalis</Emphasis> and <Emphasis Type="Italic">Rana ridibunda</Emphasis> occurring under the conditions of anthropogenic contamination of water bodies

The results of morphometric analysis of gastrulation in city populations of Rana arvalis and Rana ridibunda suggest that under the conditions of anthropogenic contamination of water bodies, the early development is more variable, if we compare less variable sizes of their eggs in the city and suburb populations. The variability of gastrulation appears to depend on the degree of contamination of spawning water bodies and is accompanied by increased correlation between the forming structures. This latter may be determined by two causes: (1) increased number of interdependent features, which leads to coordinated differentiation of structures and regulation of variability, and (2) increased coefficients of correlation within a small group of features, which leads to the formation of structures characteristic for the subsequent developmental stages, including the appearance of heterochronies.     

Comparison of variability of Rana temporaria (Amphibia, Anura) gastrula from different populations developing under the conditions of antropogenic pollution

The gastrulation of Rana temporaria embryos was studied at the natural spawning sites of Moscow city and Moscow district by means of morphometric analysis. We demonstrated that anthropogenic pollution plays an important role in morphogenesis of Anura. The measurements of embryos from the Moscow ponds made it possible to show an increased variability of gastrulation. Moreover, the level of gastrulation variability depends on the degree of pollution of a spawning pond and is coupled with increased correlations between the morphological features. This increase of correlations could be due to: (1) an increased number of correlating features (appearance of new correlations) and (2) strengthening of the existing correlations. The first way makes the gastrulation process more coordinated, and the second one causes the appearance of morphological structures, which are normally formed only at the next developmental stages.     

Comparison of variability of <Emphasis Type="Italic">Rana temporaria</Emphasis> (Amphibia,Anura) gastrula from different populations developing under the conditions of antropogenic pollution

The gastrulation of Rana temporaria embryos was studied at the natural spawning sites of Moscow city and Moscow district by means of morphometric analysis. We demonstrated that anthropogenic pollution plays an important role in morphogenesis of Anura. The measurements of embryos from the Moscow ponds made it possible to show an increased variability of gastrulation. Moreover, the level of gastrulation variability depends on the degree of pollution of a spawning pond and is coupled with increased correlations between the morphological features. This increase of correlations could be due to: (1) an increased number of correlating features (appearance of new correlations) and (2) strengthening of the existing correlations. The first way makes the gastrulation process more coordinated, and the second one causes the appearance of morphological structures, which are normally formed only at the next developmental stages.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 110–122.Original Russian Text Copyright © 2005 by Severtsova, Severtsov.     

Mechanisms of adaptive regulation of early development of Anuran amphibians dwelling the anthropogenically polluted water bodies

Comparison of the gastrulation in three Anuran species has shown the possibility of evolution of earlier morphogenesis directed to adaption to such new environmental factor as anthropogenic pollution of water bodies. Changing proportion of variability and correlation of the developing embryonic structures causes changes in the character of morphogenetic processes to the point of discoordinated development and formation of heterochronies and/or increased death rate. In the studied amphibian species, the increase of the concordent development in different populations resulted either from increased general correlation of morphogenesis or from the emergence of a group of traits with more interdependent development. Mechanisms of early development regulation comply with a single scheme, which indicates the nonspecificity of early embryogenesis reaction based on epigenetic regulation mechanisms.     

The influence of anthropogenic factors on reproduction of <Emphasis Type="Italic">Rana temporaria</Emphasis> and <Emphasis Type="Italic">Rana arvalis</Emphasis>

Studies of spawning water bodies in the city of Moscow have shown that the urban populations of common (Rana temporaria) and moor (R. arvalis) frogs are small compared with suburban populations, and their individuals lead a hidden mode of life. The recorded increase in the fecundity of females from several populations of the city of Moscow may be accompanied by an increase or the preservation of the diameter of eggs as compared with the same indicator for suburban populations. The populations in which the females produced many small eggs died out during the study period. The most prosperous populations are urban populations of brown frogs whose females spawned eggs of various sizes. We consider the formation of these clutches as a manifestation of the bet-hedging strategy compensating for mortality in adverse and unstable environmental conditions.     

The role of golf courses in maintaining genetic connectivity between common frog (Rana temporaria) populations in an urban setting

We studied population size, genetic diversity and differentiation of common frog (Rana temporaria) populations at urban golf courses and reference natural ponds in the greater Helsinki region, southern Finland. A total of 248 tadpoles from 12 locations (six golf courses, six reference sites) were genotyped with 13 polymorphic microsatellite markers. The most urban populations, situated in northern Helsinki, were the largest breeding sites having >120 (golf courses) and >200 (reference sites) spawn clumps at the time of sampling. On average, there was no difference in the number of spawns between the anthropogenic ponds at golf courses and the natural water bodies. Genetic variation within populations was substantial (H _O = 0.68) while genetic differentiation between populations was low (F _ST = 0.016; average distance = 17.6 km). The golf course populations did not differ from natural populations in terms of genetic variability or differentiation. Hence, our results suggest that golf courses contribute positively to urban amphibian populations by providing suitable water bodies for reproduction and green corridors for dispersal, thus preventing isolation and loss of genetic variability within populations.     

The correlation between the heat resistance of the zygotes of the common frog Rana temporaria and its embryos at different developmental stages

A study has been made of a correlation between the heat resistance of zygotes and embryos of the same clutches at different stages of development of Rana temporaria L. The embryos were incubated at 19 degrees C, the injurious temperature being 37 degrees C. As criterion of heat resistance served the time of the injurious temperature action which leads by the ++stra of cleavage to a 50 per cent elimination of embryos (LD50). The correlation in question has been evaluated by the rank correlation coefficient (rho). The correlation between the heat resistance of zygotes and embryos at the start of gastrulation (stage 11) was moderate (rho = 0.47). At one of neurulation stages (stage 22) this correlation was weak (rho = 0.207), to become rather high just before the cleavage (stage 28, rho = 0.63). It is assumed that the thermal selection of zygotes, i.e. of the organism at the cellular stage of development, may be oriented to result in the survival of embryos, which just before the cleavage will show both elevated heat resistance and high heritability of this character.     

Induced symmetry breaking in the egg of the frog Rana pipiens

Abstract. Unfertilized, unactivated eggs of the frog Rana pipiens were incubated in 10% Ringer solution containing 1.64 μ M 12-O-tetradecanoyl phorbol acetate (TPA). Under these conditions, the initially radially symmetric cells formed, within a few hours, a line of equatorial demarcation (IDL) which resembled the dorsal lip observed in fertilized eggs during gastrulation. Many IDLs went on to form structures which resembled lateral and ventral lips. The formation of the IDL broke the symmetry of the egg, converting it from a radially symmetric to a bilaterally symmetric object. It is suggested that such an increase in cellular organization is the result of a chemical reaction occurring in a far-from-equilibrium system of the class known as dissipative structures. It is assumed that TPA triggers this reaction as a result of its ability to stimulate protein kinase C.     

High Altitude Frogs (Rana kukonoris) Adopt a Diversified Bethedging Strategy in the Face of Environmental Unpredictability

Environmental unpredictability can influence strategies of maternal investment among eggs within a clutch. Models predict that breeding females should adopt a diversified bet-hedging strategy in unpredictable environments, but empirical field evidence from Asia is scarce. Here we tested this hypothesis by exploring spatial patterns in egg size along an altitudinal gradient in a frog species(Rana kukunoris) inhabiting the Tibetan Plateau. Within-clutch variability in egg size increased as the environment became variable(e.g., lower mean monthly temperature and mean monthly rainfall at higher altitudes), and populations in environments with more unpredictable rainfall produced eggs that were smaller and more variable in size. We provide support for a diversified bet-hedging strategy in high-altitude environments, which experience dynamic weather patterns and therefore are of unpredictable environmental quality. This strategy may be an adaptive response to lower environmental quality and higher unpredictable environmental variance. Such a strategy should increase the likelihood of breeding success and maximize maternal lifetime fitness by producing offspring that are adapted to current environmental conditions. We speculate that in high-altitude environments prone to physical disturbance, breeding females are unable to consistently produce the optimal egg size due to physiological constraints imposed by environmental conditions(e.g., duration of the active season, food availability). Species and populations whose breeding strategies are adapted to cope with uncertain environmental conditions by adjusting offspring size and therefore quality show a remarkable degree of ability to cope with future climatic changes.     

Patterns and consequences of behavioural responses to predators and parasites in Rana tadpoles

We examined the interactive effects of predators and trematodes on Rana sybatica and Rana clamitans larvae. We hypothesized that exposure to predators would increase tadpole susceptibility to trematode infection, by reducing tadpole activity and thereby increasing time spent on the bottom. We further hypothesized that the tadpoles would experience reduced rates of growth and development in the presence of either predators or parasites, and that predator presence would stimulate both species to develop larger tails and smaller bodies. Rana clamilans and R. sybatica reduced their activity in the presence of both predators and trematode cercariae. In the longer running R. clamitans experiment, predator-exposed tadpoles developed significantly shallower tails and wider bodies, while trematode infection had no effect on growth, development, or shape. Most significantly, we found that extended exposure to caged fish predators made R. clamitans tadpoles more susceptible to trematode infection. A possible mechanism for this increased vulnerability is that reduced activity in the presence of predators increases tadpoles' proximity to cercariae. Our study suggests that various factors that decrease tadpole activity–predator presence, trematode cercariae and certain pesticides–may act synergistically to negatively impact tadpole populations.     

Environmental and population dependency of genetic variability-fitness correlations in Rana temporaria

Abstract Considerable effort has been invested in studying the relationship between fitness and genetic variability. While evidence exists both for and against positive genetic variability-fitness correlations (GFC), the possible environment and population-dependency of GFCs has seldom been tested. We investigated GFCs in common frog (Rana temporaria) tadpoles reared under different temperatures and feeding regimes in four replicate populations. Genetic variability in eight microsatellite loci in 238 parents was used to estimate heterozygosity (H) and mean expected d2 in 158-sibships (4515 offspring). Generalized linear mixed model analyses of offspring fitness traits (survival to metamorphosis, developmental and growth rate) revealed that offspring survival probability was positively correlated with H, and that relationships were similar in all four populations tested. However, significant interaction between other genetic variability measures (d2, relatedness) and treatment conditions indicated that GFCs were detectable in some, but not in all environments. Interestingly, GFCs between survival and both heterozygosity and relatedness were most pronounced in stressful environments (i.e. limited food). Developmental and growth rates were significantly associated with d2 but less with H and relatedness. Furthermore, many of these GFCs were population-specific. These results suggest--in line with the contention that expression of inbreeding depression can be environment dependent--that GFCs can also be highly sensitive to the environmental conditions under which they are measured. The results further suggest that the observed positive correlation between H and survival probability is likely to be explainable by the 'general', rather than by the 'local' or 'direct' effect hypotheses.     

Molecular phenotyping of maternally mediated parallel adaptive divergence within Rana arvalis and Rana temporaria

When similar selection acts on the same traits in multiple species or populations, parallel evolution can result in similar phenotypic changes, yet the underlying molecular architecture of parallel phenotypic divergence can be variable. Maternal effects can influence evolution at ecological timescales and facilitate local adaptation, but their contribution to parallel adaptive divergence is unclear. In this study, we (i) tested for variation in embryonic acid tolerance in a common garden experiment and (ii) used molecular phenotyping of egg coats to investigate the molecular basis of maternally mediated parallel adaptive divergence in two amphibian species (Rana arvalis and Rana temporaria). Our results on three R. arvalis and two R. temporaria populations show that adaptive divergence in embryonic acid tolerance is mediated via maternally derived egg coats in both species. We find extensive polymorphism in egg jelly coat glycoproteins within both species and that acid‐tolerant clutches have more negatively charged egg jelly – indicating that the glycosylation status of the jelly coat proteins is under divergent selection in acidified environments, likely due to its impact on jelly water balance. Overall, these data provide evidence for parallel mechanisms of adaptive divergence in two species. Our study highlights the importance of studying intraspecific molecular variation in egg coats and, specifically, their glycoproteins, to increase understanding of underlying forces maintaining variation in jelly coats.     

The karyosphere during late oogenesis in Rana ridibunda.

The organization of the nucleus in the oocytes of Rana ridibunda was examined during late diplotene at the light and electron microscopic level. At this stage the chromosomes are relatively condensed and assembled in the centre of the nucleus, constituting a karyosphere. The chromosomes here are associated with the central "protein sphere" (15--20 microns in diameter), obviously at their telomeres. Numerous nucleoli are accumulated around the chromosomes, forming a karyosphere capsule and contain segregated fibrillar and granular components; structures resembling perinucleolar chromatin and fibrillar bodies (spherules) are associated with the nucleoli. Granules 30 to 40 nm in diameter are seen to surround the fibrillar spherules. "Nucleolus-like bodies" consisting of granules 10 to 15 nm in diameter which are embedded in finely fibrillar material are often associated in contact with the chromosomes. The central sphere is an accumulation of annular structures similar to those of the pore complexes of the nuclear envelope. These structures are bound to the chromosome material, the "nucleolus-like bodies" and the fibrillar bodies. A participation of "nucleolus-like bodies" in the formation of the central sphere is suggested. A possible role of the nuclear protein matrix in the construction of the karyosphere elements is discussed.     

Genetic variation in relation to demography of peripheral pool frog populations (Rana lessonae)

Summary Genetic variation in an isolated northern metapopulation of the pool frog (Rana lessonae) in Sweden was compared to that of Central European populations using enzyme electrophoresis and literature data. Of the 31 loci scored, two (EST-2 andIDH-2) were polymorphic while no variation occurred in seven of the eight loci which are polymorphic in Central European populations.The heterozygosity level of the Swedish pool frogs is very low compared to that of other anuran populations, but their mean proportion of fertilized eggs within egg masses (97.5%) was not lower than in more heterozygous species, and their body size-specific fecundity did not differ from that of Polish conspecifics. The low genetic variability of the Swedish pool frogs is discussed in relation to features of the local populations such as size (N), calculated effective size (N _e ) reproductive success and probable history. It is concluded that long-term strong fluctuations in population size caused by reporductive failure in cold years have contributed more to the low genetic variability than could a single founder event due to a recent introduction by man.     

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.     

Cerebellar afferents in the frogs,Rana esculenta and Rana temporaria

Summary Afferents to the cerebellum in frogs (Rana esculenta, Rana temporaria) were studied by use of retrograde transport of horseradish peroxidase. Following injections restricted to the molecular layer of the cerebellum cell labelling was found in the contralateral inferior olive and the ventral portion of the caudal medullary raphe. Injections involving the granular layer resulted in labelling in the ventral horn of the cervical spinal cord, the caudal spinal trigeminal nucleus, the nucleus caudalis and the medial portion of the nucleus ventralis of the vestibular nerve, the inferior reticular nucleus and the nucleus of the fasciculus longitudinalis medialis. Following larger injections, which may have spread significantly into the cerebellar, secondary gustatory, trigeminal or vestibular nuclei, labelled cell bodies were also found in the nucleus ruber, nucleus solitarius, the rostral spinal trigeminal nucleus and the rostral rhombencephalic reticular formation. It is unclear whether the fibers from these latter areas innervate the cerebellum of the frog, as they do in mammals, or only reach the underlying areas. This situation emphasizes a limitation of the HRP technique when applied to small structures as is often the case in lower vertebrates.Supported by Grant Gr 276 to U. G.-C. from the Deutsche Forschungsgemeinschaft.     

泽蛙、日本林蛙、饰纹姬蛙不同地理居群的核型多样性

本文研究了温州地区的泽蛙、日本林蛙、饰纹姬蛙的核型,并分析了9个地理居群泽蛙的核型、4个地理居群日本林蛙的核型和3个地理居群饰纹姬蛙的核型。结果表明,不同地理居群的同种蛙均有相同的染色体数和核型模式。泽蛙、日本林蛙都为 2n=26,NF=52,核模式5＋8;饰纹姬蛙 2n=24,NF=48,核模式6＋6。但同一种蛙的不同地理居群之间在SM数目和顺序、次缢痕或随体的位置等都有所不同,说明不同地理居群的同种蛙的染色体具有丰富的多样性。故保护蛙品种资源多样性,不仅要从整个群体上考虑,而且要针对每个品种（或类群）进行保护。 Abstract：The karyotypes of Rana limmocharis boie,Rana j.Japonica,and Microhyla ornata from Wenzhou were studied.The karyotypes of nine populations of Rana limmocharis boie,four populations of Rana j.Japonica and three populations of Microhyla ornata from different geographical regions were compared.The results demonstrated that the same species of different geographical populations have the same amount of chromosome and karyotypic formulae. Rana limmocharis boie and Rana j.Japonica have 2n=26,NF=52 and 5＋8 karyotypic formulae.Microhyla ornata has 2n=24,NF=48 and 6＋6 karyotypic formulae.But some dissimilarities were found among them.First,the number and sequence of submetacentric chromosome were different among them,and then the secondary contriction （SC）or satellite （Sat）were also different.It was showed that the chromosomes of same species of different geographical population have diversities.Conservation of frog genetic diversity must be considered of not only the genetic diversity conservation of the total frog population but also that of every frog breed.     

Cell adhesion to extracellular matrix in normal Rana pipiens gastrulae and in arrested hybrid gastrulae Rana pipiens female X Rana esculenta male

Rana pipiens eggs fertilized by Rana esculenta sperm (ESC) hybrid embryos develop until gastrulation in control Rana pipiens embryos (PIP) and then show morphogenetic arrest. After arrest, ESC do not gastrulate but live for 5 days as blastula-like embryos. We studied the distribution of fibronectin (FN)-containing fibrils and integrin (INT) in PIP and ESC. There are many FN-fibrils in PIP organized in anastomosing networks radiating away from the center of individual cells and across intercellular boundaries. ESC have fewer fibrils compared to PIP. These fibrils are first located between cells in disorganized arrays. After arrest in ESC, when PIP are Stage 14 neurulae, many more FN-fibrils appear. INT-staining occurs in both embryos in similar patterns. In xenoplastic transplantations, we found that the extracellular matrix on the inner surface of the ESC blastocoel roof serves as a substratum for PIP cell migration. In an in vitro assay, we found more cell adhesion to FN-substrata in PIP than in ESC. Cell locomotion rates on FN-substrata were 1.70 +/- 0.85 microns/min for PIP but only 0.46 +/- 0.56 microns/min for ESC. We also found that the inner surface of the blastocoel roof from ESC can not promote cell adhesion and locomotion when Stage 11 fragments are used for conditioning but that Stage 14 fragments can deposit a FN-fibril-rich extracellular matrix which supports PIP mesodermal cell migration at a rate of 1.26 +/- 0.38 microns/min.     

Effects of acid stress in adult Rana pipiens

The decline in frog populations is a well-recognized worldwide phenomenon and infectious disease has been implicated as a major cause in the global decline of amphibian populations. Rana pipiens are disappearing from many habitats where they used to flourish, and environmental acidification has been considered as a possible contributor to this disappearance. We present a model that integrates the results of several experiments on the effects of acid exposure on natural resistance and mortality of adult Rana pipiens. These studies suggest that different components of the natural defense mechanisms of these frogs have different acid sensitivities. We have shown previously that exposure to pH 5.5 leads to a reduction in splenic white blood cell number, viability, and to colonization of the spleen with both Gram positive and Gram negative bacteria. In this paper we show that exposure to pH 6.0 did not affect the number or viability of splenic white blood cells but did result in colonization of the spleen by bacteria. We also show that cold exposure by itself does not cause a systemic bacterial infection in adult Rana pipiens, but acid stress following cold exposure does. The data presented in this paper provide empirical evidence to support the hypothesis that acid stress may be a contributor to the decline of Rana pipiens in the northeastern region of the United States.     

History vs. current demography: explaining the genetic population structure of the common frog (Rana temporaria)

The amount of genetic variability at neutral marker loci is expected to decrease, and the degree of genetic differentiation among populations to increase, as a negative function of effective population size. We assessed the patterns of genetic variability and differentiation at seven microsatellite loci in the common frog (Rana temporaria) in a hierarchical sampling scheme involving three regions (208-885 km apart), three subregions within regions and nine populations (5-20 km apart) within subregions, and related the variability and differentiation estimates to variation in local population size estimates. Genetic variability within local populations decreased significantly with increasing latitude, as well as with decreasing population size and regional site occupancy (proportion of censured localities occupied). The positive relationship between population size and genetic variability estimates was evident also when the effect of latitude (cf. colonization history) was accounted for. Significant genetic differentiation was found at all hierarchical levels, and the degree of population differentiation tended to increase with increasing latitude. Isolation by distance was evident especially at the regional sampling level, and its strength increased significantly towards the north in concordance with decreasing census and marker-based neighbourhood size estimates. These results are in line with the conjecture that the influence of current demographic factors can override the influence of historical factors on species population genetic structure. Further, the observed reductions in genetic variability and increased degree of population differentiation towards the north are in line with theoretical and empirical treatments suggesting that effective population sizes decline towards the periphery of a species' range.